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CyTOF® 2 Mass Cytometer

1. 0 440 670 4999 70 Makeup Gas and Nebulizer Gas Note Only perform this tuning step if either 159 e Tb Dual Counts are significantly lower than the previous well performing operating session gt 400K with 204800 pushes per reading OR o Gd Tb ratio is at 3 or above If necessary tune Makeup and Nebulizer gasses according to the following protocol 1 In Instrument Setup gt DAC Channel Setup go to Nebulizer Gas and take note of the current Nebulizer Gas value Record value in the Gas _Current_X Y worksheet in the CyTOF2 Manual Tuning Log Date Nebulizer Gas Value Optimal Make Up Gas Value Tb159 Dual Count Mass155 Dual Count 4 26 2013 f lo 0 Value to record J Set Actual Curent Vake plel olele ale 2 Open the Mass Graph window and Data Acquisition Settings window Arrange the windows so that both windows are easily accessible 71 Parameter press ESC to clear Makeup Gas Start Value 0 55 End Value 1 2 step Value Settling Timeims 2000 Pushes Reading 204200 Dual Count Start Point n pulses per push 3 Select Makeup Gas and enter the parameters as shown above In the Mass Graph window S
2. 1 Insert the cones and adaptors into the Cone Cleaning Container as described above adding 10 Citranox at each step Sonicate for 15 min Rinse with DIW i solution and then rinse with DIW 4 Repeat step 1 twice using DIW Optional For more aggressive cleaning of the cones repeat step 1 using 2 Nitric Acid 5 Air dry completely before reinstalling Note The concentration of cleaning solutions sonication times and frequency of cleaning are a guide only and can be modified for the best workflow that suits the user s needs Reinsertion of the Cones Note Always install the cones before installing the Torch Assembly Skimmer Reducer 1 Place the Skimmer Reducer on the side of the Cone Removal Tool with two magnets With a No 2 pencil coat the threads of the Skimmer Reducer with graphite This allows for easier threading of the Skimmer Reducer into the interface 2 Making sure that the Skimmer Reducer is seated flush in the interface begin to turn clockwise After several turns turn back a quarter turn to make sure mis threading has not occurred If there is no resistance while turning back then continue turning clockwise 3 Repeat step 2 until the Skimmer Reducer is firmly seated Do not over tighten 4 Detach the Cone Removal Tool from installed Skimmer Reducer Sampler 1 Attach the Skimmer Reducer to the side of the Cone Removal Tool with four magnets Seat the Sampler flush in the interface M
3. It is recommended that the instrument be located near the required electrical and gas supplies as well as the coolant supply The CyTOF 2 mass cytometer is on wheels and can be moved for service and regular maintenance It is recommended that you leave a space of at least 30 cm 12 in behind the instrument to provide adequate clearance for the vent hoses Also allow space approximately 50 cm 20 in on the right side of the instrument for access to circuit breakers Access for most service procedures is through the front of the instrument The front and rear vents of the chiller must be a minimum of 24 inches 61 cm away from walls or vertical surfaces so air flow is not restricted 26 Electrical Requirements Power to the CyTOF 2 instrument is to be delivered from two 30 A single phase 200 240 V AC 50 60 Hz dedicated electrical branch circuits Table 2 2 The electrical supply requirements and approximate power consumption of the major accessories and options are summarized in Table 2 3 If the power line is unstable fluctuates or is subject to surges additional control of the incoming power may be required 60 Hertz Operation Connections The instrument is shipped with two 400 cm line cord cables The installation kit includes two NEMA L6 30 plugs 250 V 30 A for use with two 60 Hz single phase outlets The instrument is wired for power at the time of installation 50 Hertz Operation Connections The instrument is shipped with
4. Sample capillary is not inlet Masses are not correctly calibrated gas flows are not optimal Nebulizer and Make up Detector Voltage is not optimal parts of the instrument One or more hardware are not aligned properly The glassware is not clean The interface cones are not clean One or more hardware parts of the instrument need to be replaced Nebulizer and make up gas flows are too high Make sure that the sample capillary ends at the tapered portion of the sample inlet of the nebulizer see Instrument Setup and Preparation for Plasma Start in Chapter 5 Perform mass calibration see Auto Tuning in Chapter 5 Perform gas optimization see Auto Tuning in Chapter 5 Perform current optimization see Auto Tuning in Chapter 5 Perform detector voltage optimization see Auto Tuning in Chapter 5 See Chapter 6 Maintenance for proper alignment of parts and the Auto Tuning section in Chapter 5 for optimizing signals Remove the glassware according to the instructions in Chapter 6 Maintenance and clean Remove the cones according to the instructions in Chapter 6 Maintenance and clean Inspect all accessible hardware parts if there are any signs such as damage clogging and irremovable stains that suggest the part is no longer functioning optimally replace with a new one Perform gas optimization see Auto Tuning in Chapter
5. 111 Hot surface warning sign do Hot Surface Symbol not touch Potential for personal injury Radiation hazard warning l lonizing Radiation Symbol iii Any product material or substance contained under pressure including compressed gas dissolved gas or gas liquefied by compression or refrigeration Table 7 1 Hazard Symbols This table summarizes the hazard symbols that may be observed in this manual as well warning labels on the CyTOF 2 instrument Compressed Gas Hazard General Safety Guidelines This section describes some general laboratory safety guidelines For additional information we recommend The CRC Handbook of Laboratory Safety Furr 1990 and Prudent Practices for Handling Hazardous Chemicals in Laboratories National Research Council 1981 Adherence to the following safety precautions should be maintained at all times when setting up operating and maintaining the CyTOF system e Never view the ICP torch directly without protective eyewear such as safety glasses This is a bright source of ultraviolet radiation Safety glasses with side shields will provide an extra margin of safety as well as mechanical protection for your eyes Potentially hazardous ultraviolet radiation may be emitted e ICP based instruments generate high levels of radio frequency energy within the RF power supply and the torch box The RF energy is potentially hazardous if allowed to escape Safety devices and safety interloc
6. Torch Box 100 4 70 150 9 1695 200 690 System 150 6 210 450 11 5 2250 2800 9400 Venting System Recommendations The exhaust flow rate at the instrument the ability to vent the system is dependent on the blower provided by the customer the duct length material and the number of elbows or bends used If an excessively long duct system or a system with many bends is used a stronger blower may be necessary to provide sufficient exhaust volume at the instrument Additional recommendations on the venting system include e The duct casing and venting system should be made of materials suitable for temperatures as high as 70 C 160 F and be installed to meet local building code requirements e Locate the blower as close to the discharge outlet as possible All joints on the discharge side should be airtight e Equip the outlet end of the system with a backdraft damper 31 e Take the necessary precautions to keep the exhaust outlet away from open windows or inlet vents and to extend it above the roof of the building for proper dispersal of the exhaust e Equip the exhaust end of the system with an exhaust stack to improve the overall efficiency of the system e For best efficiency make sure the length of the duct that enters into the blower is a straight length at least ten times the duct diameter An elbow entrance into the blower inlet causes a loss of efficiency e Provide make up air in the same quantity as
7. Torch Box Vent System Vent Figure 2 2 Instrument rear view schematic drawing with vent positions shown 30 Flow Rate The main 100 mm 4 in venting system must provide a flow rate of approximately 70 L sec 10 150 ft min The second 150 mm 6 in venting system must provide a flow rate of approximately 210 L sec 10 450 ft min In addition to the accuracy of 10 requirement the flow through the 100 mm 4 in duct should also be stable to within 10 both in the short term during one 5 min experiment and long term through a day We recommend a 100 mm 4 in ID torch box exhaust hose and a 150 mm 6 in ID ICP Power Supply Roughing Pump air exhaust hose The CyTOF 2 instrument is supplied with 3 m 10 ft of 100 mm 4 in and 3 m 10 ft of 150 mm 6 in flexible hoses Table 2 4 A venting system that uses a single inlet duct having a flow rate of 280 L sec 600 ft min should be divided into the two separate 100 mm 4 in and 150 mm 6 in ducts equipped with individual dampers Ensure that there is access to the dampers during installation The flow rates as measured with the hoses connected to the ducts will need to be verified and adjusted during installation of the instrument The static pressure drop caused by the CyTOF 2 system is 1 2 inches H20 200 pascals Table 2 4 Venting specifications ane Hose Diam Flow Rate Anemometer Vented Outside Lab Power mm in L s ft min _ m s ft min W BTU hr
8. 3 Open the Masses per Reading window Select pulse counts for the Y axis and set max pulse counts to a value which is approximately one third to one half of the Dual Counts value for the system 5 Click Re Run as J Select Setup gt XY setup gt Setup 7 Align the window such that both windows are visible 8 Note the Current Position for X and Y before making any changes see blue arrow in figure above 9 While observing the pulse count signal in the Masses per Reading graph change X value by steps of 3000 until signal is at its highest 10 Adjust by smaller steps if necessary 11 Repeat for the Y value 69 Dual Pulse Calibration and Detector Voltage Optimization 1 Select Tuning gt Profile right click and select New Calibration 2 Select DV Optimization in the General Parameters tab Dual Pulse Calibration is selected by 3 Default Enabling of Sub calibrations V Enable DV Optimization Enabhe Dual Pulse Calibration F Enable XY Optimization Enable Gases Current Optimization Enable QC Report Inject 500uL of Tuning Solution and click Run in the Control tab 4 When the run is finished note the Optimal DV from the Results Tab in Auto Tuning window 5 i wh In Setup Setup gt DAC Channels enter this value into Actual Current Value and click Set Actual Current Value and Save Detector Bias
9. 6 Electronics 210 L s 450 ft min 33 34 Chapter 3 Instrument Interface This chapter contains annotated figures of the CyTOF 2 instrument ED S Status Panel r Sample Introduction A gt ee System E eN a J r A i d Door Handle Front Access Door Figure 3 1 CyTOF 2 Front View 35 SAMPLE SYRINGE FLOW INJECTION VALVE SYRINGE DRIVE TO NEBULI ZER Figure 3 2 Sample Introduction System Schematic Flow Injection Valve 4 Syringe Pump Drip Tray Heat Shield f gt LUA iii HAH Figure 3 3 Sample Introduction System Drain Vessel 36 Make Up Gas Line Figure 3 5 Heater and Related Parts is 1 x m rr r ae F E d d pe FAF Aeh ea Vdd dy S44 AAA y Heater Power Cord Ball Joint Clamp 37 i nS Figure 3 7 Flow Injection Valve and Syringe Pump 38 Table 3 1 Flow Injection Valve Configuration Port Number _Color Code _Function 1 Blue Nebulizer Line 2 amp 6 Black Sample Loop 1 3 Brown Waste Overflow Line 4 amp 8 Grey Sample Loop 2 5 White Upper Syringe Line 7 N A Luer Injection Port N A Green Carrier Reservoir Line Port Number _Color Code _Function 1 N A Luer Injection Port 2 amp 6 Black Sample Loop 1 3 White Upper Syringe Line 4 amp 8 Grey Sample Loop 2 5 Brown Waste Overflow Line 7 Blue Nebulizer Line N A Green Carrier Reservoir Line Ball Joint Injector Guide Pin
10. Daily QC 62 Sample Acquisition 79 Daily Cleaning 81 Shutdown Turning Off Plasma 82 Consumables 83 CHAPTER 6 85 110 MAINTENANCE Required Materials 85 Cleaning between Samples and Prior to Plasma Shutdown 86 Maintenance of the Spray Chamber and the Torch Assembly 88 Cleaning the Load Coil 92 Removal of the Cones 93 Cleaning of the Cones 95 Reinsertion of the Cones 97 Reassembly of the Torch 98 Installation of Torch Assembly 99 Checking the Torch Alignment 101 Instrument Air Filters 103 Rotary Pumps 103 Unscheduled Maintenance 107 Procedure for Expected Power Outages 109 CHAPTER 7 111 124 SAFETY Introduction 111 General Safety Guidelines 112 Environmental Conditions 113 Electrical Safety 114 Chemical Safety 117 Pressurized Gas Safety 119 Other Hazards 122 CHAPTER 8 125 132 TROUBLESHOOTING Chapter 1 Introduction to CyTOF 2 and Mass Cytometry The CyTOF 2 mass cytometer analyzes individual cells labeled with stable heavy metal isotopes using state of the art Time of Flight Inductively Coupled Plasma mass spectrometry TOF ICP MS technology Figure 1 1 With over 120 detection channels the CyTOF 2 has the exquisite ability to simultaneously resolve multiple elemental probes per cell at high acquisition rates without the need for compensation thereby maximizing the per cell information obtained from a single sample These attributes provide r
11. and AIR lights are not green turn on the argon supply and check that the exhaust AIR level is correct 53 2 Turn on the Heater a Click gt 2tup b In the Card Cage tab of the Instrument Setup page click on Heater gt On c The Heater module will take 20 minutes to reach 195 to 200 C ard Cage RFG Controler DAC Channels Setup Ports amp Commands Setup Sampler Setup 3 While the Heater is warming up connect the Nebulizer following the steps below Assemble and Install the Nebulizer Important wear gloves to prevent finger oil contamination on the nebulizer glass 1 Unscrew Swagelok Nut from the connector at the end of the Nebulizer Gas line Nebulizer Line 54 2 Remove the Front Ferrule and the black O ring Black O ring Swagelok Nut 3 Remove the clean Nebulizer from the Nebulizer soaking container and dry the surface with Kimwipe Do not touch tip of Nebulizer with Kimwipe Excess water inside the Nebulizer should also be removed 4 Put the side arm of the Nebulizer through the Swagelok Nut Do not shake the Nebulizer 5 Push the O ring onto the side arm with a tool such as the Nebulizer cap pushing it over the hose clamp bump on the Nebulizer side arm Nebulizer 55 6 Place the ferrule over the O ring with smaller orifice facing away from the Nebulizer 7 Screw the nut of the union back together p TJ 7 mari
12. the probability of pulse 22 signal overlap is negligible and particle count is most precisely determined by simply counting the number of pulses i e Pulse Count Fig 1 16 left As particle concentration increases ion pulses begin to arrive at the detector at the same time In this situation pulse count underestimates the true ion count and integrated intensity becomes a more accurate measurement Fig 1 16 right The range of data that CyTOF collects requires collection of Dual Data which means that Pulse Count and Intensity values are collected for every channel CyTOF plots the entire data range on a single Dual Signal scale the units of which are actual counts of particles that hit the detector To achieve this two things are done First a Dual Count Coefficient is applied which converts analog Intensity into actual counts according to the following formula Counts Intensity X Dual Count Coefficient Second a dual switchover threshold is applied below which Pulse Count is used and above which counts from coefficient converted analog Intensity is used Using the dual count scale CyTOF2 quantifies bound particles per cell across a wide range of signal input gt gt sam T T C Z w wu c Time ns Time ns Figure 1 16 Impact of analyte concentration on signal measurement At low analyte concentration left pulses do not overlap Because each pulse delivers a different number of electrons to the a
13. 5 129 Possible Causes Oscillations from not reached maintained Unstable Signal or Tuning Solution Proper exhaust level is Nebulizer is damaged clogged connected properly positioned correctly in the nebulizer sample Sample capillary is not inlet Nebulizer gas line is not malfunctioned Syringe Pump has Recommended Solutions With the carrier syringe running at the normal sample introduction rate 0 045 ml min carefully remove the nebulizer from the nebulizer port with all other connections intact and check the spray with a flashlight If the spray is absent or intermittent clean or replace the nebulizer Check Nebulizer Gas connection and reconnect if necessary Make sure that the sample capillary ends at the tapered portion of the sample inlet of the nebulizer see Instrument Setup and Preparation for Plasma Start in Chapter 5 Ensure that the Syringe Pump is running properly No Signal from Sample Sample is not loaded into the sample loop Ensure the sample is injected from the syringe into the sample loop 130 Possible Causes Recommended Solutions No Signal from Sample Sample is not present Continued One or more parts of the instrument are causing the problem Sample capillary is not positioned correctly in the nebulizer sample Sample leaking from carrier line or valve It is highly recommended that users add 0 1X calibration beads with t
14. a cloud of ground state atoms Subsequent electron collisions result in ionization of the cell Thus the argon ionic beam that exits the plasma contains bursts of ionic clouds corresponding to individual cells that were introduced into the torch in aerosol form Fig 1 10 Sample aerosol from Spray Chamber Auxiliary Gas 1 0 L min Plasma Gas 17 L min Figure 1 10 Cross section of the CyTOF 2 plasma torch 15 Mass Analysis The ion beam exiting the plasma contains a heterogeneous mixture of argon ions endogenous cellular ions isotopic probe ions neutral particle and photons The beam travels through the interface region into a series of low vacuum chambers that contain ion optics to eliminate unwanted materials and the time of flight mass analyzer to separate the isotopes of interest for downstream quantification and data analysis Fig 1 11 primam 7 TOF TURBOPUMP LINER eee ION REFLECTOR Bk 3 CONE INTERFACE BACKING INTERFACE PUMP PUMP Figure 1 11 CyTOF2 ion optics The ion beam leaving the torch enters the low pressure ion optical chamber green through the 3 cone interface red High mass ions leaving the Quadropole lon Guide are directed to the Time of Flight chamber black where they are separated on the basis of mass to charge ratio and directed to the detector 16 Interface Region In order to analyze the identity and amount of isotopic probes in each cell derived cloud the hi
15. accordance with all national state provincial and local health and safety regulations and laws Pressurized Gas Safety Safe Handling of Gas Cylinders Ar gas used with CyTOF systems is normally stored in liquid argon tanks or pressurized containers Carefully use store and handle compressed gases in cylinders Gas cylinders can be hazardous if they are mishandled Argon is neither explosive nor combustible Contact the gas supplier for a MSDS containing detailed information on the potential hazards associated with the gas Symbol po iescription O Any product material or substance contained under pressure including compressed gas dissolved gas or gas liquefied by compression or refrigeration Compressed Gas Hazard Table 7 4 Compressed gas hazard symbols The following hazards are associated with pressurized containers of argon e Muscle strain e Physical injury i e from a bottle falling e Suffocation 119 Warning If liquid argon is used the gas cylinder must be fitted with an overpressure regulator which will vent the cylinder as necessary to prevent it from becoming a safety hazard The following are some general safety practices for the proper identification storage and handling of gas cylinders Legibly mark cylinders to identify their contents Use the chemical name or commercially accepted name for the gas In North America as in most countries all chemical or gas storage containers must be identif
16. are accelerated and focused by an electrostatic field defined by the potentials of the reducer and a downstream conical lens and the subsequent highly focused beam is propagated to the ion optics and mass analyzer 17 lonized cells RF load coil To Quadrupole lon Deflector Sample Y ne P Reducer Injector Skimmer Argon plasma Sampler Figure 1 12 The vacuum interface which includes the three nickel interface cones sampler red skimmer blue and reducer green Quadrupole lon Deflector The beam propagating through the reducer contains some non ionized material and photons in addition to ions If not filtered neutrals can attach to instrument components resulting in signal drift and photons that reach the detector are registered erroneously as ions To eliminate these problems the beam passes perpendicularly through an electrostatic quadrupole ion deflector which turns positively charged ions towards the downstream ion optics while neutrals and photons follow an undisturbed pathway into the turbo molecular pump RF Quadrupole lon Guide The pure ionic beam leaving the quadrupole ion deflector is dominated by low mass ions that are not of analytical interest H C O N OH CO O2 Ar ArH ArO and that are of such high abundance that they would quickly damage the detector To remove these ions the beam is focused via an Einzel lens and directed into the RF only Quadrupole lon Guide Fig 18 1 13 T
17. click on Start Plasma Tr F os FS E j Tuning eB Imre Acqueion FCS Anahi Plohimear FCS To Txt Panel Plate current A 0 000 Fiaie voltage V 0 Factual Power Level W 0 010 Power 1500 Set Stat Pama Sup Plasma OS0R2013 1299 11 PM Please note that when Initiale buton is pressed Detector Voltage Liner Bias and Mirror will all be setto When water in Nebulizer is gone go back to Nebulizer Gas in DAC Channels and set value to SANPLEL In Tot 0570 0520 mmm T 59 4 When plasma starts the software will give you the following message and the status panel left is updated see following graphic oir ae 5 Click OK and allow plasma to warm up for 15 30 minutes Overview of the Software Interface and Fluidic System This section provides a brief overview of the software interface and fluidic system Software Interface Monitor Cytobank About 60 Fluidic System The CyTOF 2 utilizes a syringe pump connected to a dual loop system for sample introduction Two sample loops 1 and 2 are connected to a single sample line through a flow injection valve Once plasma has been lit the syringe pump continuously pushes carrier fluid DIW into the active sample loop as indicated by the software see red box in figure below When a new sample is loaded it will fill the idle loop and be held there until the operator clicks either the Run or Preview button When ei
18. create an unsafe environment in the proximity of electrical connections Warning If this equipment is used in a manner not specified by DVS Sciences Inc the protection provided by the equipment may be compromised Warning Lethal voltages are present at certain areas within the instrument Installation and internal maintenance of the instrument should be performed only by a DVS Sciences field service engineer or similarly authorized and trained by DVS personal e When the instrument is connected to line power opening instrument covers is likely to expose live parts e High voltages can still be present even when the power switch is in the off position e Disengage the circuit breakers before performing any service or maintenance on the cones or torch 114 Warning Before performing any maintenance turn off the RF generator power supply and allow for 2 minutes of cool down prior to accessing the ICP torch load coil and cones Warning Prior to disengaging the torch box from the vacuum chamber switch off the RF generator power using the breaker located at the left rear of the CyTOF instrument and at the right rear of the CyTOF 2 instrument Capacitors inside the instrument may still be charged even if the instrument has been disconnected from all voltage sources The instrument must be correctly connected to a suitable electrical supply see Preparing your Laboratory for the CyTOF 2 Mass Cytometer for further details F
19. the Sampler 94 5 Remove the Sampler cone from the Cone Removal Tool being careful not to come in contact with the Sampler orifice Skimmer Reducer 1 Using the other side of the Cone Removal Tool two magnets line up the pins with the two holes of the Skimmer Reducer Turn the Cone Removal Tool counter clockwise until the Skimmer Reducer comes free Remove the Skimmer Reducer from the cone removal tool being careful not to touch the orifice Cleaning of the Cones During routine maintenance and cleaning inspect the shape of the orifice and for deposits around the orifices of the Sampler and Skimmer Reducer cones Important Note During all steps of the cleaning process care should be taken that nothing comes in contact with the orifices of the cones The cones should be stacked inside the Cone Cleaning Container using the included adaptors as shown in the table below 95 Table 6 2 Adaptors and Cones Place Bottom Adapter inside the cone cleaning container container not shown Step 1 Step 2 Remove Sampler O ring then place Sampler Cone on top of Bottom Adapter Top Adaptor Place Top Adaptor on top of the Sampler Cone Cones stacked inside Cleaning Container Step 3 To prevent the O ring screws on the Skimmer Step 4 Place the Skimmer Cone on top of the Top Adapter from coming in contact with the Citranox do not fill above the level of the
20. two 380 cm line cord cables It is up to the service person installing the instrument to wire the cables with the appropriate plugs The single phase connectors must be supplied by the customer Connections to a three phase power Connection to a three phase power may be required by local electrical code The instrument can be connected to two phases and to the ground wire of the three phase line The three phase plugs must be supplied by the customer 2 Table 2 2 CyTOF 2 Instrument Power Specifications Power Consumption Maximum Volt Amperes total both circuits 9000 VA Maximum Continuous Current per circuit 20A Voltage Specification Operating Voltage 200 240 V AC Maximum Allowable Percent Sag 5 Maximum allowable Percent Swell 5 Phase singe or three Single or between two of the three phases Frequency Specifications Operating Frequency 50 or 60 Hertz Allowable Frequency Range 1Hz Waveform Specification Maximum Supply Voltage Total Distortion 5 Maximum Supply Voltage Distortion by Single Harmonic 3 Table 2 3 Electrical Requirements of Accessories Equipment Voltage AC Power Chiller Plugged into CyTOF Plugged into CyTOF Autosampler optional 100 240 V 100 VA Computer 100 240 V 1050 VA Argon Gas Requirements Argon is used as the ICP torch gas with the CyTOF 2 system The quality criteria for argon are listed below Purity gt 99 996 Oxygen lt 5 ppm Hydrogen lt 1ppm Nitroge
21. 1990 9 Sax N ed Dangerous Properties of Industrial Materials 7th ed Van Nostrand Reinhold New York USA 1989 10 Bretherick L ed Hazards in the Chemical Laboratory 3rd ed Royal Society of Chemistry London UK 1981 11 Wald P H and Stave G M eds Physical and Biological Hazards of the Workplace 2nd ed Wiley 2001 123 124 Chapter 8 Troubleshooting The following table presents recommended solutions for symptoms you may encounter If additional help is required contact technical support at support dvssciences com or by phone 1 855 387 2986 Possible Causes Recommended Solutions Plasma does not ignite RFG Circuit Breaker is Switch the circuit breaker on plasma flickers switched off achieved VGAUGE 1 must be lt 1E 6 Torr and VGAUGE2 must be 6 3E 4 Torr when plasma is not lit If these are not met a shutdown and restart of the instrument is required Contact DVS Vacuum Levels are not Check the Monitor window Sciences Technical Support for guidance incorrectly set there is around 100psi and CyTOF regulator pressure not enough argon behind the instrument to around 50 psi Also check level of argon in the tank and replace Argon pressure is Verify and adjust argon pressure on tank to tank if necessary that there may be a problem with the exhaust Exhaust is out If the EXHAUST LED light is off it indicates fan within the building 125 Symptom Possible Caus
22. Assembly off the Torch Box pins to access the Torch Body 3 Carefully grasp the Torch with one hand and firmly hold the Torch Assembly with the other hand Twist and pull the torch until it is free of the Torch holder 91 4 Clean the Spray Chamber Injector and Torch Body as detailed in Table 7 1 5 Let glassware dry completely before reassembly Cleaning the Load Coil 1 Install the Load Coil Core a _ 2 Using a Scotch Brite Ultra fine Hand Pad moistened with ultrapure methanol gently rub the surfaces of the load coil to remove any deposits 3 Remove the Load Coil Core Gently clean in between the coils with the hand pad and methanol being careful not to bend the coils 92 Removal of the Cones Note The Torch Assembly should be removed before removing the cones The cone removal tool contains magnets and pins that allow controlled removal of the sampler and skimmer reducer Side for Sampler Side for Skimmer Cone Removal Tool Removal Reducer Removal Sampler Cone 1 The Sampler cone face has four holes The two without threads which lie closer to the Sampler orifice are used for removal as G gt Sa AS 2 lt _ 2 Line up the pins on the cone removal tool with the non threaded holes being careful not to touch the orifice 93 3 Rotate the cone removal tool while pulling forward to release the Sampler from the vacuum 4 Remove the O ring from
23. DVS seiences Wipes dij lla lin Minny 7 CyTOF2 Mass Cytometer User Manual This is a Class A device and is for use in commercial industrial or business environments Warning This is a Class A product In a domestic environment this product may cause radio interference in which case the user may be required to take adequate measures CyTOF and MaxPar are registered trademarks of DVS Sciences Inc All Products and company names mentioned herein may be trademarks of their respective owners Revision 3 September 2013 DVS Sciences Inc 2013 Corporate Headquarters 639 N Pastoria Ave Sunnyvale CA 94085 2917 1 855 387 2986 Canada 70 Esna Park Dr Unit 12 Markham ON L3R 6E7 www dvssciences com Preface This manual provides e An overview of the CyTOF 2 instrument and technology e Instructions for calibration operation data acquisition and maintenance e Troubleshooting recommendations e Safety recommendations for operation of the instrument This document contains information proprietary and confidential to DVS Sciences Inc and is for customer use in the operation and maintenance of CyTOF equipment or is for vendor use in the specification fabrication and manufacture of DVS designed component parts Any other use disclosure or reproduction of the information contained herein is strictly forbidden except as DVS Sciences may authorize in writing Equipment described in this document may be protec
24. Digitizer lonic Cloud Detector gt 80 amu Figure 1 14 Separation of ions in the TOF chamber lonic clouds are subjected to an electrostatic force that orthogonally accelerates the incoming ions toward the detector As a result the ions separate based on their mass charge ratio with lighter elements reaching the detector first The cylindrical beam exiting the lon Guide first passes through the DC Quadrupole Doublet which flattens the beam so that it can enter through the rectangular entrance slit into the accelerator chamber of the TOF analyzer maintained at 10 Torr by the TOF turbo molecular pump At 13 us intervals frequency of 76 8 kHz a pulse of several hundred volts is applied to the push out plate accelerating the accumulated packet of ions orthogonally toward the reflector which redirects the ions toward the detector The electric fields in the accelerator and reflector are configured to focus ions of into tight time resolved bands regardless of initial position or energy The relationship between time of ion flight to the detector and their m z is Im el i Vz in which t and A are derived from the mass calibration procedure Because the isotopes used for probes in mass cytometry have the same charge each packet of ions resolves into a series of bands with the lightest probes reaching the detector first and each successively heavier mass reaching the detector at a later time interval Each time resolved ban
25. Go to the Analysis Parameters Tab and enter 150 for Maximum Cell Length ba Acquizibon Templates rm Hare Description Bapor AS Cate Template Created trom Analyte Table PO areshytes Created from Anahde Tabla ons abis fireatodinm Anvlyt Tabe 3 analytes Created from Anahte Table 34 anaheles Crested from Anahte Table 40 anaie Crested from Anshis Table 38 Anahe Created from Anahite Table 5 Anahies Created from nahle Table P Acquadtion Parameters Analysis Paraenabers Analyas Parameters Sonal to Arabs Dusi Count Calibeation Anahnis Mote Fladuction hhenaty Daa haiumani Fi Do analele Outaut Cell Conwetulions Pulse coum Daia Stet Puls D On The Fy Lower Convolution Threshold 200 Dua Dieta End Pulse 2 Passt Accquasilioe P pct Cel Subtraction perchannel 0 intensity ULpp SO i Dual Cami 5 1 Min Cal Lengh 10 uel Cari Sart Max Cal Length 150 Threshold Filtering Sgm J Target Cels Unlimited F 0 D amp Default Filtering Custam Exnresson Fiering 7 Enter the number of events you wish to collect in Target Cells Unlimited if 0 If you do not wish to set the number of cells to be acquired and instead run for a specified time you must enter 0 in the Found Cells Limit box Use the default settings for other parameters in the Analysis Parameters Tab Note Some settings in this tab can also be found in the Analysis tab in the Acquisition window outside of the Acquisition Templates window It is re
26. Parameters DV Parameters Gases Curent Parameters XY Parameters GC Parameters Control Results 6 After clicking Run the flow injection valve will switch and the other loop will be available for anew sample 7 Inject another 500uL into the injection port Once the first loop is finished the second loop of tuning solution will automatically be acquired for the Auto Tuning process to continue 8 A progress log will appear in the control screen as below 65 9 When calibration finishes successfully click OK Profies General Parameters Massea Parameter Dual Pulse Parameters Detector V Parameters Gases and Curent Parameters XY Parameters GC Parameters Cort Remus Retin Ain So 30082013 1200 54 PM Mass Cabbeation has enced Calibraten has finshed successfully ae nran Optimal D V found 1887 769 20556 PH Optimal D V Opt Mass 156 925 factual dual siope 020563 urent Gases 30 05 2013 12 06 07 PM Optimizing Starting syringe S05 2013 120657 PM Gas Itrochoction S mag y Oa 10 In the Results Tab ensure the 1 gt 3T b mean dual value is at least 400K and that the RSD is less than 3 Note RSD is relative standard deviation and is equivalent to CV If not perform XY alignment manually see Manual Tuning section and then repeat the Auto Tuning process from step 1 Maman Masa Tise of Plaga A Mes Tira of Agh T Resiuhgn gt
27. Tuning as described in the following steps Mass Calibration 1 Inject Tuning Solution 2 Select Tuning gt Profile right click and select New Calibration Set Current Calibration use results Set Default Calibration use parameters for new Eun Calibration 67 3 Inthe General Parameters tab de select all tuning parameters except for Dual Calibration which is selected by default ey Tuning Pats Tuning QC Beads Profes General Parmneters Massas Faramatars Dual Pues Parwneters Detector W Farursten Gants and Camera Farenetera XY Pasuessten OC Fammetwa Contd Aesuta T Sip the p and eih bampa koop migrans or the feet bere 4 Select Control tab click Run Note Running with no parameters selected will activate only Mass Calibration and Dual Pulse Calibration ears XY Alignment 1 If needed inject another 500uL of Tuning Solution 2 Set up the data acquisition parameters in the Data Acquisition Settings window t a Parameter is Reading by default if empty b Set Pushes Reading to 76 800 to allow 1 reading per second c Enter an End Value long enough for the alignment to complete An End Value of 200 acquisition time of 100 seconds is usually sufficient Parameter press ESC to cen Start Value End Value 100 Sep Value 1 Setting Timeims 2 Pushes Reading 76800 Gua Count Saat Point fn paises par push Oi 68
28. W for each loop Click Preview again Repeat steps 1 4 if background signal has not returned to baseline 81 Shutdown Turning Off Plasma 1 In Setup a Ey fa RS i Tina Bara bg hcgueheon MD dase Fa Anshai BD ip T gt oo PCI Te T Paral erp aeruphe lnc i EAE aj bate snes yt 80 0520 mm ST Peel CE Mal Perom Saup o E e THTe Cari Cage FFG Controfer DAC Channels Pots Commands Setup Sampler Setup MAB Setup APG Setup XY Setup Acquistion System Purty AS Correction Chiter Setup Plami Menegement Piste curet A 0000 Piste volage V 0 Factual Power Lewel W 000 Power 1300 Sorting Marra Gai te 1000 T eee 2 Wait until the Plasma Stop Sequence has been completed successfully message appears see below The Syringe Pump Chiller and Heater will automatically be turned off when the Plasma Stop Sequence is completed o x Plasma Stop Sequence has been completed successfully Please remove nebulizer 3 Remove the Sample Capillary from Nebulizer and then the Nebulizer from the Nebulizer Port 4 Disconnect the Nebulizer from the gas line 5 Using the syringe plus tubing tool slowly pull 10 Contrad or Decon 90 into the Nebulizer and soak for 15 min 6 Rinse the Nebulizer 2 to 3 times with DIW using the syringe and tubing tool Note When pushing liquid out of the Nebulizer only apply enough pressure so that residual liquid drips from t
29. Whee Sear rtenaty ar Wm SP se a7 ct ae Tan yen 5 6 7 080 125 ns PLASMA MODE 0 97169 o Note Values in the Monitor window are actual DETECTORVOLTAGE 1898 389 MAKEUPGAS 767 287 NEBULIZERGAS 246 3 values exactly the optimal values displayed readings Some of these may not match the Set HEATERCONTROLTMP 189 242 708 VGAUGE 1a 4 BE 7 VGAUGEZs J2E 2 42835 66 12 If settings are changed they can be restored by selecting the Tuning Profile and right clicking to choose Set Current Calibration use results Rims mi Tung GC Beads Profies Gener Pacmnaters Masses Paraasera Cus Pulse Parameters Civ Parameters Game Carent Parente XY Parameter GC Parameters Canto Aena m ma me aa e e ma ma a a i it ee eel ee me meme Set Current Calibration use results TI parals ce Calrurr TE POT PHP Run Calibration This will set any values optimized during the calibration run i e Dual Slope Detector voltage Gas Settings etc 13 Record pertinent values from the Results tab including Resolution Dual slope values for Cs and Tm Mean Dual Count Tb value RSD Dual values for Tb Cs La Tm Ir Mean 155Gd Dual counts DAC Channels settings Detector Voltage Nebulizer Gas Makeup Gas Current gt OQ00 w Manual Tuning If Auto Tuning is unsuccessful due to reasons that cannot be resolved by changing appropriate parameters proceed to perform Manual
30. a hose nebulizer clamp bump sidearm O ring front ferrule union body Schematic of the Nebulizer side arm 8 Connect Sample Capillary tube to Nebulizer a Loosen the Flangeless Nut on the connector of the Sample Capillary 56 b C Insert the Sample Capillary tubing into the sample inlet end of the Nebulizer and push up to tapered portion of the glass AGINA inside the Nebulizer Tighten the Flangeless Nut Nebulizer Port _ Port a h WN hy gt Flangeless Nut 57 d Sample Inlet of i the Nebulizer _ Tapered portion of the glass capillary Flangeless Nut Schematic of sample capillary connected to nebulizer Insert the Nebulizer into the Nebulizer Port attached to spray chamber until it reaches hard stop point 58 Optional Removing Excess Water from the Nebulizer Remove Nebulizer from Nebulizer Port In Setup gt DAC Channels find Nebulizer Gas and note setting Click Set Actual Current Value This will start the flow of Nebulizer Gas 2 fF O and click Set Actual Current Value This will turn off the Nebulizer Gas flow 4 Set the Nebulizer Gas back to the original setting and Click Save 6 Insert the Nebulizer in the Nebulizer Port until it reaches a hard stop Plasma Start 1 Check the Status Panel lights and ensure that the Argon light is green 2 Fill Carrier reservoir and empty Waste 3 In Instrument setup gt RFG Controller
31. adequate ventilation to outside air may constitute a health hazard Extreme care should be taken to vent exhaust gases properly Warning CyTOF instrument is designed for analysis of fixed permealized non live cells only Under normal operation cells are completely combusted in the ICP High levels of UV radiation inside the torch box are significantly above the lethal levels for most of single airborne cells However in the event of plasma shutdown the undigested portion of a sample can enter the torch box exhaust gases Extreme care should be taken to vent exhaust gases properly 113 Electrical Safety The CyTOF series products have been designed to protect the operator from potential electrical hazards The following section describes recommended electrical safety guidelines Symbols sd Title S scription This sign indicates high electricity electric shock Electrical machines and or Electric Shock Hazard Symbol equipment in the vicinity You may suffer severe injuries or even death The earth groud symbol represents the any terminal which is intended for connection Earth Ground Symbol to an external conductor for protection against electric shock or the terminal of a protective earth Table 7 2 Electrical Hazard Symbols This table represents the symbols you will see on the CyTOF 2 instrument and its accessories Water lines should be located away from electrical connections Condensation and potential leaks may
32. ake half a turn clockwise while applying gentle forward pressure 3 Detach the Cone Removal Tool from the installed Sampler 4 Press gently along outer edges of Sampler to make sure it is seated firmly 97 Reassembly of the Torch Note Always install the cones before installing the Torch Assembly Install the Torch Body over the two O rings of the Torch Holder by pushing and turning Turn the Torch Body so that the gas ports are oriented on top i i Connect the Auxiliary Gas line to the port closest to the Torch Holder This port is slightly angled Connect the Plasma Gas line with ignition pin to the second port This port is straight Ensure that both connections are tight Install the Ball Joint Injector by pushing and turning until it is fully inserted oe oS If the Torch and Injector are correctly installed the Injector should be 1 5 2 mm from the end of the inner portion of the Torch 2 mm distance B y 5 E s as r en a i i 98 Installation of the Torch Assembly 1 With the CyTOF door closed slide the Torch Assembly onto the Heater Box pins and push flush making sure to line up the High Voltage Connector with its port 2 When installing the Torch Assembly ensure that both screws are rotated in unison 3 Note that the screws have an internal ratcheting system on the black knobs Over a small range these knobs are free to rotate without the brass screw being turned Ther
33. and check the spray with a flashlight If the spray is absent or intermittent clean or replace the nebulizer Masses are incorrectly Perform mass calibration see Autotuning calibrated 127 Possible Causes Symptom Recommended Solutions No signal detected The analytes are not during performance selected correctly check Continued Check your analytes table and make sure the analytes of interest are selected Follow the corresponding recommended solution for the cause Nebulizer is not connected properly Check Nebulizer Gas connection and reconnect if necessary Syringe Pump has malfunctioned Signals Tb signals lt 400 000 dual counts Low Tuning solution per picogram One of the above causes Follow the corresponding recommended solution for the cause the correct temperature Heater is not on set to at 200 C check for moisture in the glassware and if necessary remove to dry after shutting off Ensure heater temperature is at 200 C If not plasma Argon Pressure is not maintained pressure is maintained 100 psi on tank and Ensure steady argon supply and proper argon 50 psi on regulator See above for recommended solutions for plasma ignition stability issues 128 Symptom Possible Causes Recommended Solutions Signals Tb signals lt 400 000 dual counts per picogram Low Tuning solution Continued positioned correctly in the nebulizer sample
34. ashing Solution to run for 2 5 minutes Repeat for the other loop Repeat for both loops with DIW Allow DIW to run for 2 5 minutes before proceeding 1 To clean the loops after beads are run inject 1mL of DIW and click Preview in the control tab This will display 10 snapshots of any ion signal traces that are detected Allow DIW to run through loop for 2 to 5 minutes and click Preview to check for residual beads To clean the second loop inject 1mL of DIW and click Preview again Allow DIW to run through loop for 2 to 5 minutes and click Preview to check for residual beads If the beads are persistent in the loops inject 500uL of Washing Solution and click Preview Allow Washing Solution to run for 2 to 5 minutes and then repeat for the second loop Run DIW for 2 5 minutes through each loop after running Washing Solution Click Preview to check status before proceeding 78 Sample Acquisition Sample Preparation Please refer to DVS protocols for sample preparation Before Acquisition It is strongly recommended that users add diluted CyTOF Calibration beads to samples as an internal standard 1 Vigorously shake the bottle with Calibration Beads Then dilute the Calibration Beads 1 10 in deionized water 2 Add the diluted Calibration Beads directly into the vial with the pelleted sample and mix well This will be the sample for acquisition 3 Normalization of cell data after ac
35. clonization iapphedio a wpiuss 8 Unfors Megative Distribution Ci Damian Deeribation Sie H Ganson Negev Hal Zer Randinization appes oip lo zest walues blaf Zerp Pandomizstion Sgm Data Type Assi mre Penin I View in Piae in Source FCS file batch mode is Target bt file FCS To Txt egan 2 bee eo T 6 Gow Aceh E PerPo tegil Mrinal Scaling 2 Toram E Commpetibie mth Fipwja a Edit Asik Ase Y Cher Took Please load file Set parameters for analysis View data in bivariate plot Perform clustering of data Convert FCS file to Text format 47 Button E Parel Solara Software view reflects status of panel on front of CyTOF 2 i View settings 1508 120 0 power detector voltage Make Monitor HEATEROONTROLTMP 187340 705 Up and VGAUGE la 4 54 7 131531 Nebulizer VGAUGE 23 3 256 22 42929 Gas heater temperature and vacuum Launch Cytobank website 48 DVS Sciences CyToF instrument Control Version 6 0 508 Login as User Copyright 2008 2013 DVS Sciences Inc a oa or Service ere ee 7 o RFG Management is Of software s version Service Table 4 2 Tuning Mode Toolbar am Da Choose ata template and Acquisition aani p i o set Settings amva E tote ite parameters in Seo Vous 1 f Sting Twa 10 Tuning mode Pues Rasing TERDI Dg aui Sua Por
36. commended to make changes in the Acquisition Templates window to ensure that the parameters are consistent across multiple samples in the same experiment 8 Settings are saved automatically once you navigate away Click on Select Template to exit this view and return to starting sample acquisitions 9 Inject 500 ul of your sample into the injection port and click Run in the Control tab 10 Once the acquisition finishes observe the data in Plotviewer if desired 80 Daily Cleaning Cleaning During Operation Cleaning between samples 1 Zi Push 1 3 mL of MilliQ or equivalent water DIW through the Sample Loop Click Preview Leave for 2 5 minutes while DIW from the carrier reservoir is running through the loop Repeat for the other loop Check background signal using Preview a If background signal has returned to baseline proceed to the next sample b If background signal is high inject 1 mL of Washing Solution into the Sample Loop and click Preview c Repeat for the second loop Allow Washing Solution to run for at least one minute and monitor with Preview e Flush DIW through both loops before proceeding to the next sample Cleaning between different users or experiments and at end of the day SS a Push 1 mL of Washing Solution through the Sample Loop Click Preview and let run for 2 5 minutes Repeat Steps 1 amp 2 for the second loop Repeat Steps 1 amp 2 with 1 mL of DI
37. d of ions of mass M is separated from its M 1 neighbor by 20 25 ns f A After the first packet of ions is pushed out and detected a second pulse pushes out the next packet of ions for detection and the cycle repeats until data acquisition is complete 20 Vacuum system The mass analysis system requires high vacuum to prevent random collisions of ions with gas molecules as they travel to the detector As described in the various sections above the CyTOF2 employs a 5 stage differential pumping system to sequentially drop the pressure from 760 Torr outside the interface to 10 Torr in the TOF chamber Table 1 1 The system includes the interface pump for the Sampler Skimmer chambers a three stage turbo molecular pump for the Skimmer Reducer chamber stage 1 the Deflector chamber stage 2 and the lon Guide chamber stage 3 and the TOF turbo molecular pump for the TOF chamber Under standard conditions the 5 stage vacuum system of the CyTOF 2 instrument operates at the five pressure ranges detailed in the table below Table 1 1 CyTOF2 vacuum system Vacuum Pump Interface Stage 1 25 L s Turbo molecular 3 Stage Stage 2 300 L s Stage 3 400 L s Turbo molecular TOF Chamber Sampler Skimmer Skimmer Reducer Deflector lon Guide TOF Pressure Torr pas 2 4 x 10 ee le 25410 0 3 1 5 x 10 21 p Data Acquisition This section describes the process whereby the ions organized by mass in the TOF cha
38. ding recommended during performance solution for the cause check 126 Symptom Possible Causes Recommended Solutions No signal detected Carrier reservoir is empty Fill the carrier reservoir with Millipore grade during performance deionized water check Continued Syringe Pump is not on Ensure that the Syringe Pump is running it is indicated by the green color on the syringe Status bar with air capillary from the nebulizer and then place the capillary into a vial Click on the Sample Intro button in the software and enter 0 3 for flow rate Wait until carrier solution replaces the air in the syringe Return the sample intro flow rate to 0 045 before reconnecting the sample Carrier Syringe is filled Purge the air First disconnect the sample capillary to the nebulizer clogged and observe the droplets emerging from the capillary If the droplets are not uniform Sample capillary is Remove the sample capillary from the nebulizer replace the capillary positioned correctly in tapered portion of the sample inlet of the Sample capillary is not Make sure that the sample capillary ends at the the nebulizer sample nebulizer see Instrument Setup and inlet Preparation for Plasma Start in Chapter 5 Nebulizer is With the carrier syringe running at the normal damaged clogged sample introduction rate 0 045 ml min carefully remove the nebulizer from the nebulizer port with all other connections intact
39. e Montor Beads Pass About ee 0s Speed 0 045 SAMPLE Inj Tot 0107 0520 amy 2 3 In the Data Acquisition Settings window follows Parameter press ESC to clear v Start Value End Value Step Value Settling Time ms Pushes Reading Dual Count Start Point in pulses per push Parameter is Reading by default if empty Set Pushes Reading to 204 800 Note Since there are 76 800 pushes per second 204 800 pushes equals 2 67 seconds per reading Open the Masses per Reading window J and select Dual counts for the Y axis and set max pulse counts to an appropriate value for your instrument ert Click Run Li Wait until the signal stabilizes and observe Tbh and Mass 155 Gd Dual count values a if Tb dual count levels are comparable to the levels in a well performing operating session gt 400K with 204800 pushes per reading AND b Gd Tb ratio is below 3 and comparable to the level from previous days with good performance begin Auto Tuning or Manual Tuning 63 c Ifthe signals are below specification adjust XY alignment manually see Manual Tuning gt XY Alignment section below and begin Auto Tuning or Manual Tuning Auto Tuning Note If any of the settings are different than described below Administrators may need to access the service mode temporarily to change these settings If applicable see note for Service Access in the follow
40. e fumes are inhaled Burns may not be immediately painful or visible Contact with eyes could result in blindness Do not use a glass beaker when working with HF as HF will attack the glass Warning Before using hydrofluoric acid you should be thoroughly familiar with its hazards and safe handling practices Observe the manufacturer s recommendations for use storage and disposal 121 Warning For better control of contamination dedicate laboratory reagents and consumables to use with CyTOF instrument and MAXPAR reagent only Other Hazards Protection from Ultraviolet Radiation and Heat Warning The plasma generates high intensity ultraviolet radiation A safety interlock is used to automatically shut off the plasma if the chamber and interface are not fully coupled Do not defeat the interlock Do not remove the shield which protects the sample introduction system the shield is designed to block any residual amounts of the ultraviolet radiation gt Protection from Radio Frequency radiation Warning Radio Frequency Radiation The instrument generates high levels of Radio Frequency RF energy which is potentially hazardous if allowed to escape The instrument is designed to contain the RF energy within the shielded enclosures of the torch compartment and the RF power supply Safety interlocks prevent you from operating the system without all covers doors and shields in place Hot Surface Temperatures Warning Ho
41. e following protocol 1 Open the Masses per Reading window and Data Acquisition Settings windows Arrange the windows so that both windows are easily accessible Parameter press ESC cew Dual Count Start Point in pulses per push 2 Select Dual count for the Y axis and set the maximum count for the Y axis to an appropriate value for the instrument 3 Select Current and enter the parameters as shown above 74 4 Inject 500uL of tuning solution and click Run 5 Choose the Current value for which the Tb Dual Count is at maximum 6 Enter this Current value in the DAC Channel setup under Instrument Setup Click on Set Actual Current Value and Save 75 When Manual Tuning is Complete 1 Save a 30 second reading file for Masses per Reading Graph using the Tuning Solution analyte template with the following parameter settings Parameter press ESC to clear Start Value End Value 30 Step Value Setting Timeime 100 Pushes Reading 204800 Dual Count Start Point Gn pulses perpush 0 1 The Tb dual counts should be gt 400 000 2 Record the following values in the CyTOF2 QC Log From the DAC Channels Tab a Makeup Gas b Nebulizer Gas c Current From the Setup gt X Y Setup gt Setup a Current X Y Values From the Active Auto T
42. e summarizes the chemical hazard symbols that you may encounter working with CyTOF reagents When handling any chemical the following safe handling guidelines should be strictly observed e Use store and dispose of chemicals in accordance with the manufacturer s recommendations and regulations applicable to the locality state province and or country e When preparing chemical solutions always work in a fume hood that is suitable for the chemicals you are using e Conduct sample preparation away from the instrument to minimize corrosion and contamination e Clean up spills immediately using the appropriate equipment and supplies and follow the appropriate MSDS guidelines e Do not put open containers of solvent near the instrument e Store solvents in an approved cabinet with the appropriate ventilation away from the instrument Warning Some chemicals used with this instrument may be hazardous or may become hazardous after completion of an analysis 117 Warning Venting for fumes and disposal of waste must be in accordance with all national state provincial and local health and safety regulations and laws e Wear the appropriate personal protective equipment PPE at all times while handling chemicals Use safety glasses with side shields goggles or full face shields according to the types of chemicals you will be handling Warning Wear suitable protective clothing including gloves specifically resistant to the ch
43. ed detector thus measures a mass spectrum F that represents the identity and quantity of each isotopic probe on a per cell basis Data is generated in fcs format G and analyzed in third party software programs H A schematic of the instrument is shown in Fig 1 3 divided by color to indicate the major steps of mass cytometry workflow Each of these steps is described in detail in the following section a aa ara a Uae ea 5 fanin ia Pratt LINER O i ILLI i a E aes TURBOPUMP INLET NEBULIZER GAS TURBOPUMP Q Pa N INTERFACE PUMP MAKE UP Figure 1 3 CyTOF2 schematic Mass Cytometry workflow is divided into sample introduction blue ionization yellow mass analysis green and data acquisition red Sample Introduction The sample introduction system de solvates the liquid sample suspension and introduces cells one at a time into the ICP source for ionization Fig 1 4 The liquid sample is introduced manually via syringe or automatically via Autosampler into a nebulizer where it is aerosolized into a heated spray chamber Within the spray chamber the high temperature partially vaporizes the aerosol and gas flows direct the aerosolized cells to the ICP source These steps are described in detail below Nebulizer Heated Spray Chamber Aerosolization Partial vaporization and delivery to ICP source Cell suspension Cell aerosol Figure 1 4 Sample Introduction The liquid sample suspe
44. efore when installing removing the Torch Assembly always ensure that the knobs are moving in the same direction as the screw Carefully preventing the knobs from accidentally rotating in the opposite direction can help ensure synchronized motion of the two screws during installation removal 99 4 When installing the torch holder tighten the screws until an audible click is heard This is to ensure that the torch holder installed properly in its end position Troubleshooting Installation of the Torch Assembly 1 In the event that the torch holder appears to be stuck on the instrument during installation removal inspect the relative position of the screw assembly with respect to one another 2 Open the front door of the instrument to be able to visualize the screw assembly from the inside 3 Compare the amount of thread engagement between the two screw assemblies the brass pieces shown below then loosen or tighten the corresponding screws to equalize the thread engagement 100 4 Turn the screws in sync to install remove torch holder as required Checking the Torch Alignment This process is necessary for two reasons Firstly it must be determined that the Torch is centered in the Load Coil Secondly the position of the Torch relative to the Interface the Z Alignment must be checked The distance from the Torch to the Interface needs to be correct so that ion clouds can travel optimally into the Co
45. elect Dual Count for the Y axis and set the maximum count for the Y axis to an appropriate value for the instrument 5 Inject 500uL of tuning solution and click Run Pian 2 Massis Per Reading Dugu Ale Mane Dump To File 6 Select the Makeup Gas value at which Tb Dual Count is at maximum when ratio of Mass Gd Th is lt 3 7 Record this value in the Gas Flow Optimization Log worksheet in a CyTOF QC Log File in the following format Date Nebulizer Gas Value Optimal Make Up Gas Value Tb159 Dual Count Mass155 Dual Count Mass155 Tb159 4 26 2013 0 15 0 82 620998 6780 1 091790956 8 Repeat this process for Makeup Gas for different Nebulizer Gas values from 0 02 of initial set point up to 0 06 For example with an initial set point of 0 15 ramp Makeup Gas at Nebulizer Gas settings of 0 15 0 17 0 19 0 21 72 9 Fillin the data obtained at each Nebulizer Gas setting for Tb159 and Mass155 dual counts at each Optimal Makeup Gas value Note If the 159 Tb Dual Count is comparable to the result from the day before and the ratio of Gd Tb is lower than 3 after ramping Makeup Gas at existing Nebulizer Gas you do not need to ramp again with different Nebulizer Gas settings Note It is not necessary to lower Nebulizer Gas for the ramping because over time the Nebulizer nozzle expands and it is unusual that lower Nebulizer Gas will give higher performance Nebulizer Gas Opti
46. emicals being handled Warning Wear protective clothing and gloves Some reagents are readily absorbed through the skin Drain Vessel A drain vessel is supplied with the CyTOF 2 system The vessel is made of HDPE and is used to gather the effluent from the Flow Injection Valve of the sample introduction system For safe operation of your system you should properly install and maintain the drain vessel and drain tubing Waste disposal procedures must be in accordance with all national state provincial and local health and safety regulations and laws Drain vessels may contain flammable acidic caustic or organic solutions cells debris and small amounts of the elements analyzed Warning It is necessary to follow appropriate waste segregation guidelines in order to prevent effluents from Warning Never place the vessel in an enclosed cabinet Doing SO may result in a build up of hazardous gases 118 Warning Do not use a glass drain vessel A glass drain vessel may break and spill toxic or corrosive liquids e Place the drain vessel in an area that is visible to the operator who can observe the level of collected effluent and empty the vessel when necessary e Check the condition of the drain tubing regularly to monitor deterioration Organic solvents deteriorate the tubing more quickly than aqueous solutions When the tubing becomes brittle or cracked replace it Empty the drain bottle regularly Disposal of waste must be in
47. ere stress during transportation Warning The radio frequency RF power supply driving the plasma torch provides up to 1 6 kW The resulting voltages may cause extensive burns even death Under no circumstances should you attempt any physical adjustments of the plasma torch when it is operating The instrument must be operated with the RF generator in the locked position at all times AN Warning Do not attempt to defeat the safety interlocks This would place the operator s safety at risk All interlocks must be engaged before you ignite the plasma 116 Chemical Safety In this section we have provided some general safety practices that you should observe when working with any chemicals The responsible individuals must take the necessary precautions to ensure that the surrounding workplace is safe and that instrument operators are not exposed to hazardous levels of toxic substances When working with any chemicals refer to the applicable Material Safety Data Sheets MSDS provided by the manufacturer or supplier symbol i eseription O Very hazardous to health when inhaled swallowed or when they come in contact Poison Hazard Symbol with the skin May even lead to death Hazardous materials toxic or very toxic materials Potential personal injury hazard Includes caustic and Corrosive Materials Hazard acid materials that can destroy the skin and eat through metals Table 7 3 Chemical hazard symbols This tabl
48. es Recommended Solutions Plasma does not ignite Chiller is not turned on The chiller should turn on automatically within plasma flickers 20 seconds after user confirms plasma start If Continued CHILL LED light is off it indicates that the chiller has not been turned on by the software Manually switch on the chiller in the Card Cage tab and ensure the CHILL LED light comes on There is moisture in Inspect glassware for moisture that may be glassware present and interfering with plasma ignition Completely dry glassware especially the Spray Chamber Injector and Torch Connections of gas line Ensure tight and correction gas line connection are incorrect on Nebulizer Soray chamber and Torch Torch has melted Argon flow is not maintained check for leaks in Torch assembly and gas lines near the interface area Check argon pressure Check Load Coil for deposits Replace Torch and if necessary Load Coil Sample capillary is not Make sure that the sample capillary ends at the positioned correctly in tapered portion of the sample inlet of the the nebulizer sample nebulizer see Instrument Setup and inlet Preparation for Plasma Start in Chapter 5 The Load Coil is not Clean the load coil so that the surface is smooth clean has micro and free of debris If necessary such as when punctures spikes there are small punctures present replace the load coil No signal detected One of the above causes Follow the correspon
49. esearchers with an unparalleled ability to generate high resolution phenotypic and functional profiles of cells from normal and diseased states Figure 1 1 The CyTOF 2 Mass Cytometer Principles of Mass Cytometry Mass cytometry employs elemental tags that have higher molecular weights than those elements that are naturally abundant in biological systems The CyTOF 2 is specifically designed to measure these high mass elemental tags on a per cell basis Cells stained with metal conjugated probes in a single cell suspension are introduced into the CyTOF 2 The cells undergo a multi step process within the instrument resulting in generation of a file that records the identity and amount of each probe on each cell Figure 1 2 m an ABCDE Cell1 43527 AECE gt Cell 16353 gt Element C re k M ad n bA T Element A Figure 1 2 Mass Cytometry Workflow A liquid sample containing cells labeled with heavy metal isotope conjugated probes A is introduced into the nebulizer B where it is aerosolized The aerosol droplets are directed into the ICP torch C where the cells are vaporized atomized and ionized Low mass ions are removed in the RF Quadrupole lon Guide D resulting in a cloud of ions enriched for the probe isotopes The ion cloud then enters the Time of Flight TOF chamber E where the probes are separated on the basis of their mass to charge ratio as they accelerate towards the detector The time resolv
50. gh temperature ion beam exiting the plasma at atmospheric pressure 760 Torr passes through a series of ion focusing and separating chambers These require low vacuum to eliminate any collisions with gas molecules on the pathway to the detector To achieve this the plasma is sampled through an interface region that dramatically reduces the temperature and pressure of the incoming ionic clouds Fig 1 12 The purpose of the interface region is to efficiently transport ions from the high temperature plasma at atmospheric pressure to the room temperature chambers that house the ion optics at less than 10 Torr The CyTOF2 uses a three cone interface to transport the ionic beam into a low pressure vacuum sampler 1 1 mm diameter orifice skimmer 1 mm and reducer 1 2 mm All three cones are made of nickel and the interface housing is water cooled to dissipate the significant heat generated by the plasma The rapidly expanding ionic clouds exiting the plasma enter the sampler cone orifice into the sampler skimmer region which is pumped by a 40 m h rotary pump to 2 3 2 5 Torr The ions then pass through the skimmer cone to the skimmer reducer region which is pumped by the 25 L s stage of the 3 stage turbo molecular pump to 2 4x10 Torr Finally the ions pass through the reducer cone which serves not only to reduce the pressure 300 L s stage of the 3 stage pump de pressurizes the chamber to 3 5x10 Torr The ions that emerge from the reducer cone
51. h the side arm and sample inlet to rinse the Nebulizer with DIW Repeat the rinse 2 3 times 12 Leave the Nebulizer soaking in DIW until next use Maintenance of the Spray Chamber and the Torch Assembly Allow heater to cool for at least 30 min after plasma shutdown before attempting disassembly Removal of the Spray Chamber 1 Slide the Heat Shield off the Heater 2 Remove the ball joint clamp which secures the Spray Chamber to the Injector 3 Open the Heater lid 4 Loosen the Make Up gas line from the Spray Chamber sidearm Nebulizer Port aN Heat Shield ey a A A Spray Chamber t 4 B Make Up Gas Connector 5 Remove the Spray Chamber from the Heater and remove the Nebulizer Port 6 Slide the entire Heater module off the guide pins and then rest on the pins below the drip tray 88 7 Remove the Ball Joint Injector by gently pulling and turning until it comes loose from the Torch Assembly 89 Disassembly of the Torch Body WARNING Before proceeding to step 1 below switch OFF the RF generator power using the breaker located at the right rear of the CyTOF2 instrument Wait at least 5 minutes for residual A electrical charge to dissipate Additional time is required to allow the ICP torch cones and the load coil to reach room temperature 1 Loosen the two thumb screws at the front of the Torch Assembly making sure to loosen in unison 90 2 Slide the Torch
52. hand thread fittings are used for fuel gas tank connections whereas right hand fittings are used for oxidant and Support gas connections e Arrange gas hoses away from foot traffic to avoid damage e Perform periodic gas leak tests by applying a soap solution to all joints and seals Exhaust Ventilation The CyTOF 2 instrument generates heat and argon gas during operation These must be exhausted from the system Exhaust venting is important for the following four reasons e To protect laboratory personnel from ozone and hot argon generated in plasma e To minimize the effects of room drafts and laboratory atmosphere on ICP torch stability 29 To help protect the instrument from corrosive vapors that may originate from samples e To remove dissipated heat which is produced by the ICP torch ICP power supply and pump motors Vent Positions The CyTOF 2 instrument has two separate vents both of which are located at the back of the instrument Figure 2 2 The Torch Box Vent exhausts plasma and the vacuum pump system and removes fumes and vapors from the torch housing It is 9 7 cm 3 8 in from the right side of the instrument when viewed from the rear and 110 6 cm 43 in above the floor The System Vent exhausts heat from the blower that cools the roughing pumps system power supply and ICP generator It is 68 cm 26 8 in from the left side of the instrument when viewed from the rear and 34 6 cm 13 6 in above the floor
53. he Nebulizer tip Do not use enough force to form a steady stream 7 Leave the Nebulizer submerged in a DIW bath prior to next use 82 Consumables Spare Parts The CyTOF 2 instrument comes with 1 spare of each part listed below The suggested total number of each spare part to have available is indicated in red Spares Additional Spares Spare Part Cat Included __Recommended Nebulizer 101794 1 2 Torch Body 101792 1 1 Ball Joint Injector 101542 1 1 Spray Chamber 105545 1 1 Sample Capillary Assembly 101519 1 1 Load Coil 105398 1 0 Sample Pump Tubing Kit 101935 1 1 Skimmer Reducer Assembly 101802 0 1 Sampler Cone 105197 0 1 Nebulizer Arm O rings 101817 0 1 pack of 5 Nebulizer Arm Ferrule 101933 0 1 pack of 5 To order additional parts visit the DVS web catalog http www dvssciences com product catalog metal php and click on the CyTOF Reagents and Spare Parts tab Reagents and Labware e Henke Sass Wolf 1ml and 3ml sterile NormJect luer syringes available from various vendors including Chem Glass Life Sciences Henke Sass Wolfe Gmbh and Agro Weber rubber free Falcon 5ml polypropylene tube with cell strainer cap 35 um catalog 352235 Calcium and Magnesium free PBS available from various vendors High grade 18Mohm De ionized water DIW e g Milli Q from Millipore Glassware and plastics polypropylene or Pyrex is recommended rather than glass to minimize Lead contamination Avoid contact with de
54. he concentration of Before the acquisition wash the sample once wash once again with DIW signals is one of markers the experiment ideally with the cell type of used interest The source of streaky Make sure the antibodies are titrated prior to 132
55. he four rods of the quadrupole are supplied with alternating current AC with opposing pairs of rods always having the same AC charge that alternates based on the radio frequency setting Low mass ions m z lt 80 gyrate dramatically and are ejected from the central path of the quadropole while high mass ions are focused ie guided through this pathway For optimal mass filtration performance the lon Guide chamber is pumped by the 400 L s stage of the 3 stage turbo molecular pump to 2 5 x 10 Torr As a result a stream of burst events corresponding to individual cells that contain only the high molecular weight isotopic probes exits the RF Quadrupole lon Guide From Quadrupole To TOF analyzer lon Deflector y lonic Cloud lonic Cloud RF Quadrupole lon Guide T Figure 1 13 The Quadrupole lon Guide removes unwanted low molecular weight argon and endogenous cellular ions from the beam that emerges from the quadrupole lon Deflector transmitting clouds that contain isotopic probe ions gt 80 amu to the TOF analyzer Time of Flight Mass Analyzer The burst event ion clouds that exit the lon Guide consist of a mixture of high molecular weight probes in a randomly distributed array These ions are then sent to the orthogonal acceleration reflectron Time of Flight TOF mass analyzer which separates the probe ions on the basis of the mass to charge ratio Fig 1 14 19 TOF Analyzer es a a 4 From lon Guide npa
56. he sample as an internal standard Refer to Product Insert for usage instructions If the beads are present but the cells are not it indicates the absence of cells in the sample itself If both the beads and the cells are not visible to the CyTOF there could be problems with one or more parts of the instrument that need to be addressed before continuing acquisition see below Refer to No Signal detected during performance check for possible causes and recommended solutions Make sure that the sample capillary ends at the tapered portion of the sample inlet of the nebulizer see Instrument Setup and inlet Preparation for Plasma Start in Chapter 5 With leakage the capillary is often too far in and has bent Trim capillary or replace if too damaged Cells are indistinct from Concentration is likely too Immediately stop the acquisition when there each other streaky high are more than 3 continuous refreshes of signals streaky signals to prevent detector damage Look for the marker s that produces this continuous streak of signals 131 Possible Causes Recommended Solutions Cells are indistinct from Too many cells are Dilute the sample with DIW Concentration of each other streaky introduced cells introduced should be 1E6 mL at signals maximum Lower cell concentrations improve Continued signal resolution intercalator is too high more with DIW If the signals are still too strong T
57. heduled Maintenance Replacement of Load Coil If the load coil shape is warped or if any deposits or damage exist the load coil needs to be replaced 1 Remove the Torch Assembly 2 Open the Front Access Door 107 3 Remove the Front Shield by undoing the clips on all 4 sides and then lifting off 4 Using a wrench loosen the 2 nuts that hold the Load Coil in place It may be necessary to apply counter force on the larger nuts ers Nuts to apply counter force 6 Install the new Load Coil Make sure that the Load Coil Core is in place before installing 108 7 Tighten nuts with the wrench Load Coil Core Nuts to tighten 8 Remove the Load Coil Core 9 Replace the Front Shield and clip in place 10 Install the Torch Assembly 11 Check that the Torch is centered in the Load Coil in section Checking the Torch Alignment above Procedure for Expected Power Outages When a power outage is scheduled for the facility the CyTOF 2 instrument needs to be properly shut down Follow the steps below to shut down prior to the power outage and restart after power is restored 109 CyTOF Shutdown 1 Ensure that the system is connected to the argon supply the vacuum chamber will be filled with argon when vented 2 Press Vacuum Off button on the side panel on the left side of the instrument below the circuit breakers 3 Wait for 10 minutes Initially the turbo pumps will be heard to be sl
58. i papusch i 49 Table 4 2 Button es cej a inia Use E Gump To Fie PEPER Logit View Mass Per intensity Reading i P pulse coun irg or dual count of selected isotopes View selected Time of Flight TOF Range or Mass Range TOF Mass Peak Continue to N A run sample from current sample loop N A to change Switch valve sample loops 50 Table 4 3 Syringe Toolbar Speed 0 045 SAMPLE2 Inj Tot 0 520 0 520 SAMPLE2 Inj Tot 0 520 0 520 Sample Loop In Use Volume of Syringe Injected Total Volume Syringe Refresh Button 51 52 Chapter 5 CyTOF 2 Operation This chapter describes daily operation of the CyTOF 2 Mass Cytometer including e Preparation and Startup pp 53 60 e Overview of the Software Interface and Fluidic System pp 60 61 e Daily QC Background Check p 62 Performance Check p 63 Auto Tuning pp 64 67 Manual Tuning pp 67 76 Bead Sensitivity Test p 77 Cleaning after Tuning p 78 Oo O O O e Sample Acquisition pp 79 80 e Daily Cleaning p 81 e Shutdown Turning off Plasma p 82 e Consumables p 83 Preparation and Startup 1 Check Status Panel lights To do so open the CyTOF Software and locate the Status Panel sPanel within the interface on the left The panel parameter indicator lights should be lit as in the figure below If the ARGON
59. ied by means of approved labels i e WHMIS labels Note See the Preparing Your Laboratory for the CyTOF 2 Mass Cytometer guide for detailed information on the correct storage of gas cylinders Handling Cylinders e Move cylinders with a suitable hand truck after ensuring that the container cap is secured and the cylinder properly fastened to the hand truck e Never roll or drag a compressed gas cylinder Use a wheel cart e Always use a stand or safety strap while using or storing a cylinder e Replace the protective cap on the valve when the cylinder is not in use Use only regulators tubing and hose connectors specifically approved by an appropriate regulatory agency to be used with the gas in the cylinder e Never lubricate regulators or fittings Do not force caps off with tools If stuck contact the supplier e Arrange gas hoses where they will not be damaged or stepped on and where objects will not be dropped on them e Do not refill gas cylinders Check the condition of pipes hoses and connectors regularly e Perform gas leak tests at all joints and seals of the gas system regularly using an approved gas leak detection solution e Close all gas cylinder valves tightly at the cylinder when the equipment is turned off Liquid Argon Handling Carefully inspect argon tanks prior to use e Ensure that good ventilation is maintained in the laboratory space that will contain the liquid argon cylinders 120 Warning Oxygen mo
60. ing sections 1 Click on Tuning ny FCS To Txt 2 Select Tuning Tab for Auto Tuning 3 Inthe Profiles Tab right click and select New Calibration Tuning Qc Beads Lier Set Current Calibration use results Set Default Calibration use parameters for mew Run Calibration 4 Under General Parameters ensure that all the desired tuning parameters are checked with proper delay timings Perform Auto tuning with DV Optimization Dual Pulse Calibration Gases Current Optimization and QC report enabled in General Parameters Mass calibration and Mass Resolution are automatically performed and calculated whenever Auto Tuning is performed as Stop the prompt and swich sample loops sutomatioglly for the first time 64 Service Access Notes 1 Note that introduction rate is pre set to 0 03 mL min in General Parameters Do not change this setting 2 Under QC parameters ensure that all of the relevant tuning analytes are selected with the correct parameters as shown in the following figures a If necessary right click or hit F4 on the Keyboard to pull up the Periodic Table to select any additional analytes b Ensure that the integration level is set to 204 800 pushes and introduction rate is 0 045 mL min is shee Turing QC Beads Proftes OC Parameters 5 Inject 500uL of Tuning Solution and click Run under the control tab Tuning GC Beads Profiles Masses
61. is exhausted by the system An airtight laboratory can cause an efficiency loss in the exhaust system e Ensure that the system is drawing properly by placing a piece of cardboard over the mouth of the vent Environmental Conditions The CyTOF 2 mass cytometer has been designed for indoor use only The environment in which the instrument is installed should meet the following conditions e Room Temperature The room temperature should be between 15 and 30 C 59 and 86 F with a maximum rate of change of 2 8 C 5 F per hour e Relative Humidity The relative humidity should be between 20 and 80 non condensing e Elevation The instrument should not be operated at an elevation greater than 2 000 m 6 500 ft above sea level Use of the instrument at elevations greater than 2 000 m is subject to acceptance by local inspection authorities The instrument should be located in an area that is e Free of smoke and corrosive fumes e Not prone to excessive vibration e Out of direct sunlight e Away from heat radiators WARNING Do not use the instrument in an area where explosion hazards may exist 32 Table 2 5 Instrument specifications summary Gas Argon 299 996 Purity 345 7kPa 80 1 psi 20 L min Coolant Filtered Glycerol and DIW 3 8 L min 345 14 kPa 50 2 psi Electrical Power Maximum Voltage 9000 VA Operating Voltage 200 240 V AC Operating Frequency 60 Hz Exhaust Vents Open 4 Torch Box 70 L s 150 ft min
62. ith a small sample of new oil Change the vacuum pump oil if it has an unusual color is dark contains particles or appears dirty or turbid Typically if the oil is the color of honey then it does not need changing If it is a darker color then it should be changed immediately The following section details the procedure for changing the oil for both pumps 103 1 Turn off the vacuum pumps with the switch on the right side of the instrument 2 After the vacuum pumps have completely shutoff open the front access door 3 Pull the lever to the left to open the bottom compartment 104 105 5 Open the top fill cap on the backing pump Bottom Oil Cap gt 7 6 Open the bottom oil cap and drain the oil into a tray or container 7 Replace the bottom oil cap Pour HE 100 type vacuum oil into the top end fill hole using a funnel until the level is full in the window 8 Replace top cap Be careful not to over tighten to prevent leaking 106 9 Open the valve on the interface pump and drain the oil into a tray or container 10 Refill the oil using a funnel Fill to approximately 3 4 full using interface pump sight glass as a guide behind hand in picture above 11 Replace the cap Be careful not to over tighten to prevent leaking 12 Close right side door and door to bottom compartment Close front access door 13 Start the vacuum pumps and wait for the vacuum level to return to specification Unsc
63. ks should not be bypassed or disconnected e The power supplies of the CyTOF instrument are capable of generating potentially lethal voltages Store the removable instrument handle separately from the instrument No maintenance should be performed by anyone other than a DVS Sciences Service Specialist or by the customer s own DVS trained and appropriately certified maintenance personnel e Do not allow smoking in the work area Smoking is a source of significant contamination as well as a potential route for ingesting harmful chemicals e When installing or moving the instrument contact a DVS Sciences field service engineer for assistance The total weight of the instrument is 295 kg 650 Ibs e Food should not be stored handled or consumed in the work area 112 Environmental Conditions Refer to the Preparing Your Laboratory for the CyTOF 2 Mass Cytometer guide for the recommended environmental conditions Laboratory Ventilation Toxic combustion products metal vapor and ozone can be generated by the CyTOF system depending upon the type of analysis Therefore an efficient ventilation system must be provided for your instrument When the plasma is on hot gases are vented through two exhaust vents located at the back of the instrument Detailed information on exhaust vents are described in the Preparing Your Laboratory for the DVS Sciences Inc CyTOF 2 Mass Cytometer guide Warning The use of CyTOF instruments without
64. led nebulizer gas Both liquid at 45 uL min and gas at 0 15 0 35 L min flows are directed towards the spray chamber through a tapered end Fig 1 8 Because the liquid chamber has a small inner diameter the sample velocity is high and pressure is low within the nebulizer and as the sample exits the tip concentric pressure exerted by the exiting nebulizer gas breaks it up into a fine droplet aerosol 12 Nebulizer Spray Chamber 200 C Liquid Cell Suspension gt s Pet gt To ICP torch 45 ul mL NebulizerGas Make up Gas 0 3 L min 0 7 L min Figure 1 7 Sample aerosolization and delivery to the ICP torch Liquid cell suspension is aerosolized by nebulizer gas as it exits the nebulizer Make up gas carries the aerosol through the heated spray chamber where it is partially de solvated and delivered to the ICP torch L Figure 1 8 Nebulizer Liquid sample enters from the left and argon Nebulizer gas from the bottom Sample chamber narrows into a capillary pulling liquid rapidly to the tip enlarged at right with liquid sample indicated in red where shear forces exerted by accelerated nebulizer gas break the liquid into aerosol droplets Spray Chamber The aerosolized sample exits the nebulizer directly into the spray chamber which is housed within a 200 C heating block Argon gas called make up gas is pumped into the spray chamber 0 7 L min and this high flow of heated gas partially vap
65. mal Makeup Mass 155 Dual Value L min Gas value L min Tb159 Dual count _ count __Mass155 Tb159 0 15 1 07 486 000 9300 1 6 0 17 1 00 540 000 13500 2 5 0 19 0 90 526 500 13700 2 6 0 21 0 80 486 000 12500 2 5 9 Using the Table just created choose the combination of Nebulizer Gas and Makeup Gas where the Tb signal is the highest as long as the Gd gt Tb ratio is below 3 In this example see figure above the optimal combination is Nebulizer Gas at 0 17 and Makeup Gas at 1 00 10 Enter the gas values in Instrument Setup gt DAC Channel Setup and click Save 11 If Gd to Tb ratio does not go lower than 3 try a new Nebulizer Card Cage RFG Cont roller DAC Channels Setup Ports amp Commands Setup Sampler Setup Actual Actual Actual Parameter Name Current Update Min Max Value RFVolts 244 41 9 130 Set Actual Current Value j Middle Point 80 8 A Set Actual Curent Valve _ Detector Bias 0 5000 Set Actual Current Value Detector Voltage 0 2300 Set Ad Curent Value DO2 40 20 i PulserP p pR Update these 2 values PulserN 0 Liner 0 Mirror Current T 5 2 ALA IY 4 x St UTET LVA 73 Current Optimization 159 Note Only perform this tuning step if the Tb Dual Counts are significantly lower than the previous well performing operating session gt 400K with 204800 pushes per reading If necessary tune Current according to th
66. mber are detected converted into digital values and analyzed Fig 1 15 lons Sorted by m z ratio in TOF chamber Element Cell1 4 3 52 7 gt epn Cell3 2 45r 9 kaniy ar ae Sy i F mF ik i HEE y pA A Ay 7 a J amp eee Element Element Dynode Detector Mass Spectrum Data analysis Figure 1 15 Detection of ions and data analysis workflow Detector The ions separated in the TOF chamber are detected using a discrete dynode electron multiplier When an ion strikes the first dynode of the detector several secondary electrons are liberated These electrons strike the next dynode where they generate more electrons This process is repeated at each dynode resulting in an electron pulse that is captured by the anode of the detector The output analog signal is amplified and converted by a dual 8 bit digitizer to digital values at 1 ns sampling intervals The digitizer trigger delay dictates the first mass channel to be recorded per push while the segment length dictates the mass range to be recorded per push Instruments are set to collect data from at least 120 mass channels each corresponding to 1 amu typically starting at mass 88 Dual Count Scale CyTOF resolves multi element samples using time of flight with ions from each isotope arriving at the detector centered in discrete 20 25 ns time windows within each 13us push depending on their mass to charge ratio At very low particle concentrations
67. ment in your laboratory The CyTOF 2 mass cytometer is shipped to you as a complete system with the exception of the following items which must be obtained prior to installation electrical power exhaust vents and argon gas supply with approved regulator When preparing the laboratory for instrument installation by a DVS Sciences Field Service Engineer the following items must be considered e System layout e Electrical requirements e Argon gas requirements e Exhaust ventilation e Environmental conditions 25 System Layout Figure 2 1 CyTOF 2 mass cytometer B and components including the chiller A computer C and monitor D The CyTOF 2 system consists of the main instrument a refrigerated chiller Polysciences cat 6105PE and a system computer with workstation Fig 2 1 The dimensions of the instrument chiller and optional autosampler are given in Table 2 1 Note that the autosampler is designed to rest on the instrument shelf and so does not occupy an additional lab space The system computer may be placed on a bench or a separate computer table Table 2 1 Dimensions of CyTOF 2 Chiller and Autosampler Component _ Width cm in Height cm in Depth cm in Weight kg Ib CyTOF 2 97 38 132 52 79 31 285 628 Chiller 38 15 64 25 67 27 81 178 Autosampler 39 16 24 10 36 14 20 44 Autosampler is optional and when installed rests on the instrument shelf and therefore does not take up any additional lab space
68. n lt 20 ppm Water lt 4ppm Argon gas at 80 1 psi 522 7 kPa can be supplied to the CyTOF 2 system from liquid or gas storage tanks The choice of liquid argon or gaseous argon tanks is determined primarily by the availability of each and the usage rate 28 Safe Handling of Gas Cylinders The permanent installation of gas supplies is the responsibility of the user and should conform to local safety and building codes The following are a list of safety precautions that should be observed when handling argon gas cylinders e Fasten all gas cylinders securely to an immovable bulkhead or a permanent wall e When gas cylinders are stored in confined areas ventilation should be adequate to prevent dangerous accumulations Move or store gas cylinders only in a vertical position with the valve cap in place e Locate gas cylinders away from heat or ignition sources including heat lamps Cylinders have a pressure relief device that will release the contents of the cylinder if the temperature exceeds 52 C 125 F e When storing cylinders external to a building the cylinders should be stored so that they are protected against temperature extremes including the direct rays of the sun and should be stored above ground on a suitable floor e Gas cylinders should be clearly marked to identify the contents and status e g full empty e Do not attempt to refill gas cylinders e Use only approved regulators and hose connectors Left
69. nes Check that the Torch is centered in the Load Coil 1 Open the CyTOF door 2 Install the Torch Positioning Tool may be either black or silver in color in the end of the Torch 101 p Va J n ffia Torch Positioning Tool n Wa 3 Turn the Torch Positioning Tool It should spin freely 4 Ifthe tool doesn t spin freely the Torch Box is not installed properly Remove the Torch Assembly and reinstall following steps 6 amp 7 in section Reassembly of the Torch above oe Check the Z Alignment 1 Gently push the Torch Positioning Tool in as far as it will go The outer edge of the Torch Positioning Tool should be flush with the edge of the torch W 102 Troubleshooting the Z Alignment Z Positioning Cap 1 Recheck the Z Alignment as described above Instrument Air Filters The CyTOF 2 is equipped with a large and small air filter on the underside of the instrument These air filters remove particles from the argon gas supply The filters can be removed by opening the bottom of the instrument and pulling them out These filters are inspected and changed by the DVS Sciences Service Engineers on an annual basis Rotary Pumps The CyTOF 2 has two rotary pumps an interface pump and a backing pump Maintenance of the vacuum pumps includes inspecting the pumps and changing the pump oil Inspect the pump oil daily and compare the appearance of the oil w
70. nitors should be installed in the laboratory to ensure that dangerous enriched oxygen environments are not created Warning Liquid argon in the cylinder is maintained at extremely low temperatures Personal protective equipment including gloves safety glasses and long sleeved clothing should be worn when operating the cylinder e Cryogenic liquid cylinders contain a vacuum that helps to maintain the integrity of the liquid argon in the cylinder This vacuum may become compromised if the following symptoms are observed o The outer vessel shows signs of frosting o The outer vessel sweats in humid conditions o The pressure relief valve opens continuously until the vessel is emptied e Never lay or store the cylinder on its side Cylinders should be stored in a vertical position e Do not roll a cryogenic liquid cylinder e Cylinders may be moved using a cart overhead crane or hoist Sample Handling and Preparation Sample preparation for the CyTOF 2 may require the handling of organic or corrosive solutions MAXPAR reagents that are used with CyTOF instruments are supplied in a solution form Please refer to the information supplied with MAXPAR reagent Material Safety Data Sheets MSDS for safe handling of the reagents Hydrofluoric Acid Trace amounts lt 0 1 w v of hydrofluoric acid HF may be present in the wash out solution Hydrofluoric acid is toxic and extremely corrosive It will readily burn skin and lung tissue if th
71. node and therefore different intensity values it is more precise to count pulses when ion concentration is very low Here the pulse count is 1 At higher analyte concentrations right pulses overlap and counting pulses will underestimate the true number of particles that hit the detector Here the pulse count is 8 if we count discernible peaks even though 16 ions hit the detector Thus at high analyte concentration it is more accurate to use integrated intensity and convert this intensity value to counts using a calibration coefficient Cell Detection and Acquisition Data File format Data for each 13us push is digitized sequentially and integrated to obtain mass peaks for the channels selected for analysis The resulting record is processed according to cell event selection criteria set by the user These criteria described in detail in Chapter 6 include a 23 minimum signal threshold and a range for event duration consistent with single cell events As a result the data acquired contains the integrated number of total ion counts for each selected analyte on a per cell basis These data are saved as text txt and flow cytometry standard fcs 3 0 format for data analysis in compatible software programs 24 Chapter 2 Preparing Your Laboratory for the CyTOF 2 Mass Cytometer This chapter is designed to help you to understand the CyTOF 2 mass cytometer instrument and conditions required for successful installation of the instru
72. nsion is syringe injected then aerosolized by the nebulizer into the spray chamber which partially vaporizes the aerosol and delivers it to the plasma Delivery of sample to the nebulizer Liquid cell suspensions are introduced into the instrument manually using a syringe or automatically using an Autosampler Manual Introduction The manual sample introduction system upstream of the nebulizer is composed of the sample syringe syringe drive flow injection valve dual sample loop system waste vessel and carrier fluid vessel Figure 1 5 First the initial sample is loaded into a 1 mL syringe and injected through the sample loading port into one 500 uL loop of tubing of the dual sample loop system During this step the flow injection valve is rotated to open a fluidic pathway from the sample syringe through the sample loop and out to the waste vessel Thus any sample in excess of 500 10 uL is lost to the waste vessel circuit Once the sample is loaded into the loop the flow injection valve rotates opening a fluidic pathway from the syringe drive to the sample loop to the nebulizer Then the syringe drive pushes carrier fluid through the fluidic circuit delivering the sample to the nebulizer The syringe drive controls the volumetric flow rate and is typically operated at 45 uL min A couple of special features of the system optimize sample throughput by minimizing time between samples First the syringe drive automatically recharge
73. ody tube and the sample injector contains argon auxiliary gas flowing at 1 L min that is used to change the position of the base of the plasma relative to the sample injector The innermost chamber inside the sample injector transmits the argon stream and sample aerosol from the spray chamber directly into the center of the plasma The torch assembly is mounted inside an induction load coil that is supplied with radio frequency generated current that creates an electromagnetic field 14 Formation of the ICP discharge and ionization of the sample Plasma the fourth state of matter consisting of charged particles is formed by collision induced ionization of argon gas within an intense electromagnetic field First argon plasma gas flows tangentially from the outer chamber of the torch body RF power supplied to the load coil produces an oscillating current 40 MHz creating a strong electromagnetic field precisely at the point the plasma gas exits the outer chamber A high voltage spark strips away free electrons from the exiting argon atoms These free electrons accelerate dramatically in the electromagnetic field and collide with sufficient energy to ionize the argon gas into plasma Temperatures within the plasma typically range from 5 000 to 10 000K When the aerosolized sample is introduced through the injector into the base of the plasma the water droplets are rapidly vaporized The de solvated individual cells are then broken down into
74. or 50 Hz installations a means of electrically grounding the instrument must be available The power supply must have a correctly installed protective conductor earth ground and must be installed or checked by a qualified electrician before connecting the instrument Warning Any interruption of the protective conductor earth ground inside or outside the instrument or disconnection of the protective conductor terminal is likely to make the instrument dangerous Connect the instrument to a correctly installed line power outlet that has a protective conductor connection earth ground Do not operate the instrument with any covers or internal parts removed Do not attempt to make internal adjustments or replacements except as directed in the user Manual Disconnect the instrument from all voltage sources before opening it for any adjustment replacement maintenance or repair Use only fuses with the required current rating and of the specified type for replacement Do not use makeshift fuses or short circuit the fuse holders If there are any signs that the instrument is no longer electrically safe for use make the instrument inoperative and secure it with a lockout against any unauthorized or unintentional operation The electrical safety of the instrument is likely to be compromised if the instrument 115 o Shows visible damage o Has been subjected to prolonged storage under unfavorable conditions o Has been subjected to sev
75. orizes the sample to minimize condensation for optimal ionization as it directs the aerosol to the ICP source 13 Ionization The mixture of single cell aerosol droplets and argon that exits the spray chamber is transmitted to the ICP source where it is successively vaporized atomized and ionized in the plasma for subsequent mass analysis Fig 1 9 The formation and characteristics of the plasma responsible for the ionization process are described below Torch RF Load Coil Plasma TELS ney To Interface Vaporization Atomization Ionization Figure 1 9 Electromagnetic energy generated by the RF load coil surrounding the quartz torch sustains argon plasma orange that vaporizes atomizes and ionizes individual cell aerosols from the spray chamber The positive ion component of the cell derived plasma cloud enters the ion optics and mass analyzer chambers of the CyTOF2 through the interface Plasma Torch The plasma is created within the plasma torch by induction using a radio frequency generated electromagnetic field The torch consists of the torch body a fused assembly of two concentric quartz tubes and a quartz sample injector tube that is inserted inside the torch body When assembled the torch consists of three concentric chambers The outermost chamber between the torch body tubes contains argon plasma gas flowing at 17 L min that is ignited to form the plasma The central chamber between the inner torch b
76. owing down gradually Then the venting valve will open and the chamber will be filled up with argon through the purge valve slowly at controlled pressure 4 After the turbo pumps have slowed down the power can be shut off At this point the reading on the vacuum gauge controller VGC402 will be in Torr range as opposed to the lt 1E 6 Torr operational pressure and 1E 6 1e 4M Torr when the turbo pumps are slowing down but the venting valve is not yet open It will open after turbo pumps are slowed down completely 5 Let flush with argon for 10 minutes 6 Switch OFF the circuit breakers in this sequence AC Outlets Backing Pump RF Generator and System 7 Leave argon supply ON if one wants to save on argon and can compromise on time taken for vacuum to build up you can turn the argon supply OFF CyTOF Startup 1 Ensure that the Argon supply was left ON during the shutdown If not then open the Argon gas supply and wait for 2 hrs 2 Switch ON the circuit breakers in this sequence System RF Generator Backing Pump amp AC Outlets 3 After the system is powered up the RFG Test LED on the instrument control panel will be red This is normal Just press RFG Test button on the side panel below the circuit breakers to turn it off 4 Press Vacuum ON button VG1 will come on first then TP1 and TP2 will come up after 6 minutes or so and finally VG2 will come on after up to gt 30 minutes 5 Chiller may have to be manually
77. quisition can be done by dividing the signal value from the marker of interest by Eu signal value in the same sample This resulting normalized signal is independent of any intrinsic variability of the instrument The Normalization tool available from your FAS can also be used Set up Acquisition Parameters and Sample Introduction 1 Torun samples open the Acquisition window from Acquisition jn the menu bar 2 Specify a pathway and filename to save an FCS file 3 Setup Acquisition Parameters a Acquisition time is the duration of the sample acquisition in seconds When the default syringe speed of 0 045 mL min is used it will take approximately 650 seconds more accurately 667s to collect the 500 uL Sample Loop volume b The acquisition delay is typically 40 sec c Detector stability delay should be set to 10 sec Acquisition Analysis Control Acquisition Files FCS Filename Display only Acquisition Parameters Acquisition time s 650 Pushes to show on a graph Acquisition delay s 40 Graph restresh interval ms 1000 Detector stability delay s 10 79 4 Right click in the Analyte table shown below to apply a template F3 or to make a new template with the periodic table 5 Save the template by selecting Create Template From F7 Apphr Template F3 Create Template From Fi Periodic Table Fal 6 The Acquisition Templates window will then open with the selected analytes saved
78. s Figure 3 8 Front View of Torch Assembly 39 3 1 Auxiliary Gas Line Plasma Gas Line 7 Plasma Gas Torch Holder Torch Body Auxiliary Gas Port gt High Voltage Connector Figure 3 9 Rear View of Torch Assembly F a a miaii Torch Body A 2 ws Figure 3 10 Interior View with Front Access Door Open 40 Figure 3 11 Torch Box 41 Table 3 3 Other CyTOF 2 Parts Circuit Breakers and Right Side of Cords Instrument Digital Readout of Vacuum Gauges Left Side of Heater Temperature Instrument Make Up Gas and Nebulizer Gas Behind Sampler Cone Skimmer Reducer Cone Table 3 4 CyTOF 2 Glassware Nebulizer Spray Chamber Ball Joint Injector Torch 43 44 Chapter 4 Software Interface Table 4 1 Main Toolbar Administrator Mode Tuning Sample Intro Acquisition FCS Analysis Piotewer FCS To Txt sPanel Monitor Start up and Pea A D O ommmamummenuan mansir f shutdown Seen ae plasma ee Access DAC Channels Perform manual XY alignment a OMoego1 12 00 11 Pu ease note Put when baida Gaon ni pipi iha Diea taga Liner had ed Meroe well a oe net fo Check instrument performance and optimize settings Run Calibration Beads Tuning Table 4 1 Button Set syringe speed Set parameters for collecting sample data Acquisition 46 CE i iiss Taget ACS Ae F Pasn
79. s with carrier fluid when it is low by drawing from the carrier fluid vessel thereby eliminating the need to manually recharge the pump Secondly the dual sample loop system allows washing of the alternate sample loop during data acquisition from sample in the first loop SAMPLE SYRINGE FLOW INJECTION VALVE SYRINGE DRIVE p Dga NA a 4 A 7 TO NEBULIZER Ti 5 SAMPLE LOOP 1 SAMPLE LOOP 2 CARRIER FLUID Figure 1 5 Schematic of Sample Introduction System upstream of the nebulizer 11 Autosampler If the CyTOF2 is connected to the Autosampler Fig 1 6 samples loaded into 96 well plates are automatically introduced into the system allowing unattended instrument operation and sample data acquisition The autosampler contains a separate dedicated liquid sampling automation system that is described in detail in the CyTOF Autosampler Manual Figure 1 6 Image of the AS 5 autosampler Delivery of de solvated sample aerosol to the ICP source For liquid sample analysis it is critical to remove as much water as possible from the sample so that it can be efficiently ionized in the plasma This is achieved first by aerosolizing the sample in the nebulizer followed by delivery of heated aerosol to the plasma by the spray chamber Fig 1 7 Nebulizer The CyTOF 2 employs a glass concentric nebulizer consisting of an inner capillary that carries the liquid sample and an outer chamber that carries argon gas flow cal
80. switched off When the system is in a stand by mode all LEDs mentioned above are lit Wait for the vacuum reading on the VGC402 to reach 1E 6 Torr before attempting to start plasma NOTE Vacuum readings can be found behind the lower door on the left side of the instrument 110 Chapter 7 Safety Introduction This document describes general practices designed to aid you in safely operating the CyTOF 2 and its accessories This advice is intended to supplement not supersede the normal safety codes in your country The information provided does not cover every safety procedure that should be practiced Ultimately maintenance of a safe laboratory environment is the responsibility of the operator and the operator s organization Please review all manuals supplied with the CyTOF 2 and accessories before you start working with the instrument to prevent personal injury or damage to the instrumentation Carefully read the safety information in this chapter and in the other manuals supplied When setting up the instrument or performing analyses or maintenance procedures strictly follow the instructions provided Symbols The warnings provided in this manual must be observed during operation and maintenance of the CyTOF 2 Symbol a i escription General warning symbol Indicates a hazardous situation that if not avoided could result in death or serious injury Warning Symbol Radio Frequency Radiation Symbol
81. t Surface Temperatures The torch components the interface and the sample introduction system components remain hot for some time after the plasma has been shut off Allow sufficient time for these items to cool to room temperature before you handle them 122 References 1 Furr K ed CRC Handbook of Laboratory Safety 3rd ed The Chemical Rubber Co Press Florida USA 1990 2 National Research Council Prudent Practices for Handling Hazardous Chemicals in Laboratories National Academy Press Washington D C USA 1981 3 Compressed Gas Association USA Safe Handling of Compressed Gases in Containers pamphlet no P 1 11 ed August 2008 4 Compressed Gas Association USA The Inert Gases Argon Nitrogen and Helium pamphlet no P 9 4 ed March 2008 5 Material Safety Data Sheets MSDS USA DIN Sicherheitsdatenblaetter genormte Formular DIN Nr 52900 FRG Product Information Sheets UK 6 Other sources of information include OSHA Occupational Safety and Health Administration United States ACGIH American Conference of Governmental Industrial Hygienists United States COSHH Control of Substances Hazardous to Health United Kingdom 7 Helrich K ed Official Methods of Analysis 15th ed Association of Official Analytical Chemists Inc Arlington VA USA 1990 8 Bretherick L Bretherick s Handbook of Reactive Chemical Hazards qin ed Butterworth amp Co Ltd London UK
82. ted under one or more patents filed in the United States Canada and other countries Additional patents are pending Software described in this document may be furnished under a license agreement It is against the law to copy the software on any medium except as specifically allowed in the license agreement Portions of this document may make reference to other manufacturers products which may contain parts that are patented and may contain parts whose names are trademarked Any such usage is intended only to designate those manufacturers products as supplied by DVS for incorporation into its equipment DVS Sciences Inc assume no responsibility or contingent liability for any use to which the purchaser may subject the equipment described herein or for any adverse circumstances arising therefrom Table of Contents PREFACE 3 4 CHAPTER 1 7 24 INTRODUCTION TO CyTOF 2 and MASS CYTOMETRY Principles of Mass Cytometry 8 Sample Introduction 10 lonization 14 Mass Analysis 16 Data Acquisition 22 CHAPTER 2 25 34 PREPARING YOUR LABORATORY FOR THE CyTOF 2 MASS CYTOMETER System Layout 26 Electrical Requirements 27 Argon Gas Requirements 28 Exhaust Ventilation 29 Environmental Conditions 32 CHAPTER 3 35 44 INSTRUMENT INTERFACE CHAPTER 4 45 52 SOFTWARE INTERFACE CHAPTER 5 53 84 CyTOF 2 OPERATION Preparation and Start Up 53 Overview of the Software Interface and Fluidic System 60
83. tergents which may be a source of Barium e General reagents should be of analytical grade 83 84 Chapter 6 Maintenance Instrument cleaning and maintenance ensures optimal operational performance of your CyTOF 2 instrument Table 6 1 summarizes routine cleaning and other required maintenance Subsequent sections will detail how these procedures are performed Table 6 1 Summary of Routine Cleaning and Maintenance Part Nebulizer Spray Chamber Injector Torch Body Cones Sampler and Skimmer Reducer Load Coil Interface Pump Oil Backing Pump Oil Air Filters Daily Weekly depending on Instrument usage Weekly Annually or as needed Every 6 months Annually Required Materials 1 Sonicator 2 Cleaning Solutions a Contrad 100 or Decon 90 Decon Labs Cat 1504 1 gallon dilute to 10 in MilliQ grade water DIW b Citranox Liquid Acid Cleaner and Detergent Alconox Cat 1801 4 x 1 gallon or Sigma Aldrich Cat Z273236 1 ea 3 7L dilute to 10 in DIW c Nitric Acid Seastar Chemicals Inc Cat S020101 dilute to 2 in DIW US Suppliers VWR BDH Aristar Ultra Nitric Acid Thermo Fisher Optima Nitric Acid Operator Operator Operator Operator Engineer once per year Operator Engineer once per year Operator Field Service Engineer 10 Contrad 100 or 10 Decon 90 Deionized Water DIW 10 Contrad 100 or 10 Decon 90 DIW 10 Citranox 2 Nitric Acid optional DIW Ul
84. ther of these buttons is clicked the valve switches and carrier fluid is pushed through the previously idle loop and data acquisition of the newly loaded sample begins The previously active loop is then idle and available for loading of another sample Selecting Re run or Re preview will not cause valve switching and so sample acquisition will continue to be from the currently active loop Users can check what loop is in use on the upper right syringe pump status bar as shown below Beads Pass About Speed 0 045 SAMPLEL Hij Tot 0 107 0 520 EE EA For optimal signal intensity and resolution the Syringe Pump speed is set at 45uL min 0 045 mL min and this defines the sample flow rate The maximum flow rate at which plasma can be sustained is 60uL min 0 060 mL min The syringe pump flow rate can be changed in the Sample Intro window 61 Daily QC The CyTOF2 should be tuned every day for optimal performance and data quality Tuning can be performed automatically or manually Check Background Open the Acquisition window Acquisition Click on the Control tab 3 Click Preview to view background signal and ensure that the sample introduction system is clean and ready for Tuning a Acquisition 62 Check Performance before Tuning 1 Ensure that the Syringe Pump speed is set to 0 045 mL min in Sample Intro or by checking the upper right portion of the software interfac
85. trapure Methanol HE 100 vacuum oil HE 100 vacuum oil N A 85 d Ultrapure Methanol 3 Tools dedicated to cleaning CyTOF parts only a Glassware brushes of varying sizes b Scotch Brite Ultra fine Hand Pad 3M 7448 Cleaning Between Samples and Prior to Plasma Shutdown Nebulizer Timely removal of the Nebulizer from the elevated temperature environment of the Heater Module as well as soaking in DIW when not in use will help to avoid clogging of the tip 1 Remove the sample capillary tubing and set aside 2 Remove the Nebulizer from the Nebulizer Adapter 3 Loosen the connection of the Nebulizer gas and remove the Nebulizer 86 4 Retighten the union being careful to retain the O ring and ferrule 5 Connect the side arm of the Nebulizer to the syringe and tubing tool With the Nebulizer tip submerged in detergent pull slowly on the syringe plunger to fill the nebulizer with detergent 7 Repeat steps 5 amp 6 with the syringe and tubing tool connected to the sample inlet 8 Soak the Nebulizer in detergent for 15 min 9 Pull back on the syringe plunger to draw air into the syringe 10 Attach the syringe and tubing tool to the nebulizer side arm and slowly push on the plunger to expel detergent from the Nebulizer into a waste beaker Note Detergent should drip out If the detergent comes out in a stream the syringe is being depressed too quickly 8 11 Use the syringe and tubing tool on bot
86. uning Profile gt Results Tab a Optimal Detector Voltage DV 76 Bead Sensitivity Test Open Acquisition specify in the directory a location to save the file and set up the experiment details such as Acquisition files and parameters as follows Acquisition Analysis Control Acquisition Files FCS Filename Display only Preserve IMD Acquistion Parameters Acquistion time s 120 Pushes to show on a graph 225 M Apply Template F3 Acquisition delay s 50 Graph resfresh interval ims 1000 Detector stability delay js 10 Periodic Table Fj Create Template From F7 Right click in the analyte table on the right to apply a template F3 or to make a new template with the periodic table and save the template by selecting Create Template From F7 Use the default settings in the Analysis Parameters Tab Inject 500uL of beads and click Run in the Control Tab Once the acquisition finishes observe the data in a third party FCS file reader Gate singlet population and doublet population Add event of singlets to event x 2 of doublets This total should be at least 12000 events If not rerun beads 151 153 Check that the mean of singlet population for Eu or Eu is at least 1000 If not rerun beads 77 Cleaning After Tuning Tuning Solution 1 PRA Beads To clean the loops after the Tuning Solution is run inject 1mL of Washing Solution and click Preview Allow W

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CyTOF® 2 Mass Cytometer