Muse™ EGFR-RTK Activation Dual Detection Kit User's Guide
Contents
1. add additional buffer to bring the volume up to 100 ul or proceed to the next sample Ifthe sample volume is greater than 100 UL then the lack of events is probably due to a clog A clog or blockage can be caused by cell aggregates cell debris bleach crystals or other particulates Perform a backtlush to flush out the Clog into a tube containing 20 bleach Then run Quick Clean to remove bleach residue If this procedure does not alleviate the problem refer to the Muse Cell Analyzer User s Guide tor additional troubleshooting tips or contact Millipore Technical Support for help 9 The Muse EGFR RTK Activation Dual Detection Kit works best with samples in single cell suspension Cell aggregates may clog or be excluded from the flow cell affecting the accuracy ot the results If you wish to use the Muse EGFR RTK Activation Dual Detection Kit with a clumpy cell line it is recommended to order Muse Cell Dispersal Reagent Catalog No MCH100107 to disaggregate the cells Contact customer service or visit our website at www milipore com muse or detailed information on the Muse Cell Dispersal Reagent and assay method For more troubleshooting tips refer to the Muse Cell Analyzer User s Guide For more information contact the Millipore office nearest you In the USA call 1 800 MILLIPORE 1 800 645 5476 Outside the USA visit our website at www millipore corv offices for up to date worldwide contact information You can al2. oral or written which are inconsistent with this warranty or such publications are not authorized and if given should not be relied upon Inthe event of a breach of the foregoing warranty EMD Millipore Corporation s sole obligation shall be to repair or replace at its option the applicable product or part thereof provided the customer notifies EMD Millipore Corporation promptly of any such breach If after exercising reasonable efforts EMD Mlilipore Corporation is unable to repair or replace the product or part then EDM Millipore shall refund to the Company all monies paid for such applicable Product EMD MILLIPORE CORPORATION SHALL NOT BE LIABLE FOR CONSEQUENTIAL INCIDENTAL SPECIAL OR ANY OTHER DAMAGES RESULTING FROM ECONOMIC LOSS OR PROPERTY DAMAGE SUSTAINED BY ANY COMPANY CUSTOMER FROM THE USE OF ITS PRODUCTS Unless otherwise stated in our catalog or other company documentation accompanying the products our products are intended for research use ony and are not to be used for any other purpose which includes but s not rmited 10 unauthorized commercial uses in viro agnostic uses e vivo or in vivo therapeutic uses or any ype of consumption or application to humans or animais 2009 2012 Merck KGaA Darmstadt AI rights reserved No part of these works may be reproduced in any form without permission in wrting Cat No MCH200102MAN October 2012 Revision A
3. 95 pL 1X Assay Buffer in a cell suspension Adjust the slider bars on the X and Y axis to place all populations Non expressing Inactivated and Activated on scale If the cell sample is not treated e g activated a great majority of the cell population will fal either in the Inactivated upper left or Non expressing quadrants ower left Adjust the quadrant markers to place the cell population s immediately to the left of the marker see diagram below Adjust the quadrant markers You can move the marker intersection in any direction as well as adjust the angle of each line To move the markers as they are touch the open circle at the intersection and drag the markers to make large changes or touch the arrow buttons below the plot to make small changes A To adjust the angle of either line touch the solid circle and drag in an arc or touch the arrow buttons below the plot B and C p Pi A 4 i Wes O I Tit EE Eo Back 7 Select Next when the marker adjustments are complete 8 Verity the settings If the settings are correct select Next Otherwise select Back and repeat steps 4 through 7 as necessary Verify Settings 9 Enter the sample ID by touching the field then using the keypad to input the ID Touch Done when you re finished entering the ID If necessary change the Events to Acquire by touching the fi
4. If there appears to be day to day variation of the staining pattem ensure the Muse Cell Analyzer is working properly Check the Muse System Check log to ensure day to day instrument variation is low References 1 Martin L A et al 2003 Enhanced estrogen receptor ER alpha ERBB2 and MAPK signal transduction pathways operate during the adaption of MCF 7 cells to long term estrogen deprivation J Biol Chem 278 33 30458 68 2 Simeonova P P et al 2002 C Sre dependent activation of the epidermal growth factor receptor and mitogen activated protein kinase pathway by arsenic Role in carcinogenesis J Biol Chem 277 4 2945 50 3 Zhang P et al 2000 Peroxynitrate targets the epidermal growth factor receptor Raf 1 and MEK independently to activate MAPK J Bio Chem 275 29 22479 86 4 Palmer H J et al 1999 Age dependent decline in mitogenic stimulation of hepatocytes Reduced association between Shc and the epidermal growth factor receptor is coupled to decreased activation of Raf and extracellular signal regulated kinases J Biol Chem 274 16 11424 30 5 Sorkin A et al 1992 Multiple autophosphorylation sites of the epidermal growth factor receptor are essential for receptor kinase activity and intemalization Contrasting significance of tyrosine 992 in the native and truncated receptors J Bio Chem 267 12 8672 8 Related Products Muse H2A X Activation Dual Detection Kit C
5. Muse EGFR RTK Activation Dual Detection Kit User s Guide Catalog No MCH200102 50 tests FOR RESEARCH USE ONLY Not for use in diagnostic procedures USA a Canada Phone 1 800 437 7500 Fax 1 951 676 9200 Australia 61 3 9839 2000 war milipore com Introduction EMD Millipore s Muse Dual Detection kits are a series of products which include a pair of antibodies that bind to the same protein one to detect total protein expression and another to detect the phosphorylated form of the same target So by using two parameter analysis we can achieve target specific detection of phosphorylation and by doing so eliminate false positives while enhancing the signal to noise ratio Data generated using the Muse Cell Analyzer with the Muse software provides Percentage of inactivated cells Percentage of activated cells via phosphorylation Percentage of non expressing cells The epidermal growth factor receptor EGFR ErbB 1 HERI in humans is the cell surface receptor for members of the epidermal growth factor family EGF tamily of extracellular protein ligands EGFR is a member of the ErbB family of receptors a subfamily of four closely related receptor tyrosine kinases EGFR ErbB 1 HER2 c neu ErbB 2 Her 3 ErbB 3 and Her 4 ErbB 4 Mutations affecting EGFR expression or activity could result in cancer EGFR exists on the cell surface and is activated by binding of its specific ligands including EGF
6. Next Run and repeat steps 9 through 12 for the remaining samples NOTE During the run a message may appear prompting you to load a tube of DI water for a Quick Clean Load the water then select Clean to perform the Quick Clean Select Next to continue with the run The frequency of Quick Cleans was set by your system administrator Your administrator may also have chosen to allow you to skip the Quick Clean when the prompt appears You can choose to perform additional Quick Cleans at any time during a run by selecting Clean in the title bar then Quick Clean from the menu 14 When you have acquired the last sample select Finish 15 Optional Select Options in the tle bar to rename the data set export the data set save the current instrument settings or view the event log Refer to the Muse Cell Analyzer User s Guide for more information 2 Quick Clean Load sample tube cortaining DI Water Results The software performs calculations and displays the data in two plots A dot plot displaying cells which are inactivated non expressing and activated on a bivariate plot indicating EGFR expression and phosphorylation for a given testing sample Abar graph illustration of the same data calculating the expression of each cell population as a percentage Results from each run are stored in a data file as well as its corresponding spreadsheet CSV file The data file and spreadsheet file contai
7. Reagent to adjust the cell warring during concentration between 300 and 700 cells pL acquisition High CVs wide peaks or false peak Although the assay procedure has been optimized to function for multiple cells types every cell line behaves differently The wide peaks or false peak may indicate that The sample is poorly fixed and stained as a result of cell aggregates Ensure your sample is a single cell suspension before fixing and staining Cell concentration is too high Decrease the number of cells by diluting the sample to 300 700 celis uL The Muse Cell Analyzer gives the most accurate data when the flaw rate is between 300 and 700 cells uL Low level of staining Although the assay procedure has been optimized to function utilizing multiple cell types every cell line behaves differently A low signal may indicate that the calls need to be stained at a higher volume Verify that the System Check procedure was performed and the results passed Variability in day to day experiments Ifthe results are inconsistent check that the samples were well mixed prior to acquisition Cells may quickly settle in your samples and your results will be inaccurate unless the cells are mixed just prior to acquisition Monitor experimental cell cultures to ensure that cell viability and cell numbers being analyzed are consistent Any drop in cell numbers or viability can influence experimental results
8. and transforming growth factor a TGFa Upon activation EGFR undergoes a transition from an inactive monomeric form to an active homodimer EGFR may also pair with another member of the ErbB receptor family such as ErbB2 Her2ineu to create an activated heterodimer There is also evidence to suggest that activated EGFRs form clusters although it remains unclear whether this clustering is important for activation itself or occurs subsequent to activation of individual dimers EGFR dimerization stimulates its intrinsic intracellular protein tyrosine kinase activity As a result autophosphorylation of five tyrosine Y residues in the C terminal domain of EGFR occurs These are Y992 1045 Y1068 Y1148 and Y1173 This autophosphorylation elicits downstream activation and signaling by several other proteins that associate with the phosphorylated tyrosines through their own phosphotyrosine binding SH2 domains These downstream signaling proteins initiate several signal transduction cascades principally the MAPK Akt and JNK pathways leading to DNA synthesis and cell proliferation Such proteins modulate phenotypes such as cell migration adhesion and proliferation The kinase domain of EGFR can also cross phosphorylate tyrosine residues of other receptors it is aggregated with and can itself be activated in that manner All Muse Dual Detection kits are optimized on the Muse Cell Analyzer Both antibodies provided in the kit are carefully titra
9. atalog No MCH200101 Muse PI3K Activation Dual Detection Kit Catalog No MCH200103 Muse MAPK Activation Dual Detection Kit Catalog No MCH200104 Muse Bol 2 Activation Dual Detection Kit Catalog No MCH200105 Muse System Check Kit Catalog No MCH100101 Muse Count amp Viability Kit 100T Catalog No MCH100102 Muse Count amp Viability Kit 600T Catalog No MCH600103 Muse Count amp Viability 200X Catalog No MCH100104 9 Muse Annexin V amp Dead Cell Kit Catalog No MCH100105 10 Muse Cell Cycle Kit Catalog No MCH100106 11 Muse Caspase 3 7 Kit Catalog No MCH100108 12 Muse MultiGaspase Kit Catalog No MCH100109 18 Muse Mitopotential Kit Catalog No MCH100110 14 Muse Cell Dispersal Reagent Catalog No MCH100107 16 Warranty EMD Millipore Corporation EMD Millipore warrants its products will meet their applicable published specifications when used in accordance with their applicable instructions for a period of one year from shipment of the products EMD MILLIPORE MAKES NO OTHER WARRANTY EXPRESSED OR IMPLIED THERE IS NO WARRANTY OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE The warranty provided herein and the data specifications and descriptions of EMD Millipore products appearing in EMD Milipore s published catalogues and product literature may not be altered except by express written agreement signed by an officer of EMD Millipore Representations
10. ave good viability prior to use 2 For certain cell cultures cell pellets may become hazy or transparent following the fixation step making it dificult to see if sampling a small collection of cells for flow analysis it is recommended that all steps be performed in a smaller collection tube e g centrifuge tube 3 Do nat mix or interchange reagents from various kit lots 4 Mix each cell sample thoroughly on a mixer before acquiring samples for consistent and accurate results However avoid vigorous mixing which can cause cellular breakdown and splashing resulting in volume loss and erroneous results 5 The default number of events to acquire is 1000 events You may select a different number however your statistical error will increase as you decrease the number of acquisition events 6 If results deviate from expected values prepare a freshly stained sample and reacquire the data 7 Periodically run Quick Clean using a tube of DI water after every 20 sample acquisitions to prevent a buildup from cellular debris in the system If your samples contain significant amounts of cellular debris run the Quick Clean cycle more often to prevent clogs or blockage 8 If you are acquiring data from a sample but the progress bar is not moving there is probably either insufficient volume to continue to acquire the sample or a blockage of the flow system First check to ensure that there is at least 100 uL of sample in the tube If not
11. commended that you run the cell samples shortly after completing the sample preparation While some cell types have been shown to yield stable results for up to 24 hours after Cell fiation permeabiization antibody staining if propery stored the stability of individual cell types may vary le considerations When dealing with phospho specific activation detection fixation of cell samples after cell reatment s is critical to capture the phosphorylation activation event Some activation state cell signaling responses are transient and may be lost if cell cultures are not fixed immediately following treatment Cell fixation permeabilization and staining will ake approximately 50 minutes Acquiring data on your Muse Cell Analyzer takes less than 3 minutes per sample depending on the cell concentration and desired number of events to acquire Always perform a System Check prior to performing the assay For details refer to the Muse Cell Analyzer User s Guide Preparation of Reagents 4 Assay Buffer Assay Buffer is supplied at 5X concentration and should be diluted to 1X with deionized water prior to use Prepared 1X Assay Buffer is stable up to one year Store at 2 8 C 2 Antibody Working Cocktail Solution The kit contains two 2 antibodies which can be used in multiplex Prior to antibody staining of cells prepare an antibody working cocktail solution by addition of the following Add 5 pL of anti phospho EGFR Tyr1173 Ale
12. eld then selecting the value from the pop up menu Select Next sample Info Sampat 001 cme e S ewmsogae neve w 10 Follow the onscreen instructions and mix the first sample Load the sample on the instrument loading arm Select Run to acquire the sample Run Sample Og ase gra rite nto bar graph Mestre enced ch w 11 When acquisition is complete the results are displayed If desired select Plots to display a dot plot and a bar graph for the sample 003 Sample_003 Serene You can view or change the sample ID as well as add annotations for the current sample by selecting the Sample Info Tab To print the results for the current sample select the printer tab n 12 Optional If changes are needed to the gates assigned touch the dot plot to enlarge it then adjust the cell size gate according as described in steps 4 and 6 respectively You cannot adjust the EOE EE L cell size threshold after the sample has been acquired It you adjust the gate on subsequent samples and wish to apply the changes to other samples that you already acquired select the Apply Changes button in the title bar Select the samples you want to apply the changes to or choose Select All then select Apply The sample you originally made changes to must be selected ize Senare aie enee 13 If no adjustments are needed select
13. gs Load the sample for adjusting the settings and select Run Note Perform the adjust settings step using a negative control e g total antibody then verity the settings using a positive control Or to retrieve previously saved instrument settings select Retrieve Settings For more information on retrieving settings see the Muse Cell Analyzer User s Guide Oy Oy Os 4 Fine tune the settings for the EGFR EXPRESSION and CELL SIZE INDEX plot if necessary on screen instruction for example Adjust the CELL SIZE INDEX slider accordingly to capture the cell population of interest see Drag the threshold let or right to exclude cell debris Drag to make large changes Touch the arrow buttons located below the plot to make small changes The arrow buttons appear after you touch the threshold function NOTE If the acquisition times out after two minutes you can select Abort to restart the adjust settings step or Next to accept the settings and continue to the next step Adjust Settings Step 112 Cape eel ppulaion or eu ace NDEN aer dust Stings Siep 1012 5 Select Next when you have completed the adjustments 6 Fine tune the settings for the EGFR EXPRESSION vs PHOSPHORYLATION plat if necessary SETTING THE GATE To set the quadrant marker properly prepare a cell sample containing only the total antibody e g 5 uL of anti EGFR PECyS antibody
14. n the following statistics Sample Number Sample ID Percent totals for the Inactivated ActivatediNon expressing cell types Mean Fluorescence Intensity for the Inactivated ActivatediNon expressing cell types Sampie inc a smes One B po fee a ees merit tex an a A Hide bp SL S E EE H Figures A and B A431 cells were exposed to 100 ngimL EGF for 5 minutes to induce an EGFR signaling cascade response fixed permeabilized and then stained with both anti phospho EGFR Tyr1173 and ant EGFR antibodies in multiplex Samples were acquired using the Muse Gell Analyzer and statistical results are shown above Figure A shows the results summary while Figure B shows results displayed by both dot plot and bar graph data The statistics captured in this assay show the relative percentages for each population as it is calculated within the total cell population Cells which express EGFR can be seen by the data on the top two quadrants of the dot plot inactivated and activated representing about 93 of the total cell population But of this cell population 91 is activated upon treatment indicating activation of the EGFR signaling pathway is present By presentation of both datasets one can now determine the total phospho ratio within their testing samples 3 Technical Tips 1 For cellular staining and analysis to be most effective make sure that test cells h
15. so view the tech service page on our website at www millipore corvtechservice Troubleshooting Potential Problem Experimental Suggestions Acquisition taking longer than expected or progress bar stops during acquisition Ensure that the System Check procedure was run and passed Ifthe progress bar stops during acquisition the fluid system may be clogged Run a Quick Clean procedure to clean the capillary It can be performed during or after an assay Instrument clogging Ithe instrument is clogged run a Quick Clean procedure to clean the capillary It can be performed at anytime during an assay between samples No detectable phosphorylation activation in testing samples Since phospho specific activation can be very quick transient responses in order to capture this phosphorylation event samples must be immediately fixed to freeze the given activation state in time Low Cell Concentration warning during Ensure that cells are counted properly prior to beginning the experiment The assay instructions are optimized to give you a range of cells between 300 700 cells yL in the final sample volume so accurate population count aagi results are obtained A substantial decrease in cell numbers can lead to difficulty in adjusting settings High Cell If the concentration of the stained cell sample Is high 1200 cells pL dilute Concentration the sample further with Muse Cell Cycle
16. ted and optimized together to ensure maximal performance when run in multiplex alleviating the need for any additional optimization This kit contains optimized fixation permeabilization and assay buffers to provide researchers with a complete solution for EGFR signaling analysis Test Principle The Muse EGFR RTK Activation Dual Detection Kit includes two directly conjugated antibodies a phospho specific anti phospho EGFR Tyr1173 Alexa Fluor 555 and an anti EGFR PECy5 conjugated antibody to measure total levels of EGFR expression This two color kit is designed to detect the extent of EGFR pathway activation by measuring EGFR phosphorylation relative to the 1 total EGFR expression in any given cell population By doing such the levels of both the total and phosphorylated protein can be measured simultaneously in the same cell resulting in a normalized and accurate measurement of EGFR activation after stimulation Moreover simultaneous measurement of both total and phospho EGFR confirms target specificity of the phosphorylation event Together a total and phospho antibody duo performed in multiplex provides an enhanced and more reliable detection of the phospho total ratio within a mixed cell population The antibody pair provided in the kit has been carefully titrated to ensure the ability to measure total and phospho EGFR simultaneously on the same protein for accurate determination of protein level and activation S
17. tely measuring 10 1000 ul Tabletop centrifuge capable of achieving 300 x g Mechanical vortex Deionized Water for butter dilution Cells of interest in suspension e g Jurkat Microcentrifuge tubes with screw caps 1 5 mL VWR Catalog No 16466 030 or equivalent 9 Muse Cell Analyzer 10 Muse System Check Kit Catalog No MCH100101 11 Guava ICF Instrument Cleaning Fluid Catalog No 4200 0140 optional Precautions The instructions provided have been designed to optimize the kit s performance Deviation from the kit s instructions may result in suboptimal performance and may produce inaccurate data Some assay components included in the kit may be harmful Please refer to the MSDS sheet for specific information on hazardous materials MSDS forms can be found on the web page or by contacting Millipore technical services During storage and shipment the directly conjugated antibodies may condense within the vial For maximum recovery of the product centrifuge original vial prior to removing cap The conjugated antibody is light sensitive and must be stored in the dark at 2 8 Do not use reagents beyond the expiration date of the kit Storage Al reagents must be stored at 2 8 C All kit components are stable up to four 4 months from date of receipt if stored and handled correctly Please avoid repeated changes affect the integrity of the product Before You Begin Itis highly re
18. the flasks in a 7 incubator with 5 CO2 Exposure time and treatment concentrations are determined at the discretion of the end user 4 Deactivate cells by exchanging out the growth media with fresh growth media or 1X PBS All cell treatments and experimental samples are determined by the end user This section is provided only as an example for inducing an EGFR response for measurement of phospho EGFR activation 1 Fix and Permeat ize Cells 5 Alter cellular deactivation spin down the treated and untreated testing samples at 300 x g for 5 minutes and discard the media 6 Resuspend cells by adding 500 ul of 1X Assay Butter per one milion cells for larger cell samples i e 5x10 cells add 2 5 mL 1X Assay Buffer to cell sample Essentially add 50 pL of 1X Assay Buffer for every 100 000 cells evaluated 7 Add equal parts Fixation Buffer to cell suspension 1 1 So for every 500 pL of 1X Assay Buffer per one milion cells add an additional 500 yl Fixation Buffer for a total of 1 mL cell fixation solution and mix sample by gently pipetting up and down Similarly add 50 iL of Fixation Buffer for every extra 100 000 cells evaluated to keep the 1 1 ratio consistent Incubate for minutes on ice 8 Spin down cells at 300 x g for 5 minutes in a tabletop centrifuge and discard supernatant 9 Permeabilize cells by adding 1 mL ice cold Permeabilzation Buffer per one million cells and incubate on ice for 5 min
19. ufficient reagents are provided to perform 50 tests Detailed assay instructions are included to assist in analysis and to ensure the correct cell concentration is obtained during acquisition of sample data For Research Use Only Not for use in diagnostic procedures x EGFR Signaling Pathway The EGFR signaling pathway is activated upon stimulation of the EGFR receptor with EGF resulting in the activation of many downstream effectors EGFR activation has a direct impact on the STAT PISK Akt and MAPK signaling pathways all of which result to multiple biological processes such as cell survival cell growth apoptosis and cell proliferation Summary of Protocol Prepac cutee tor ipernantaon et 0p o arty Cennkge cats at 300 x g tor Smin aret iow nuba ior 30min scaure samples on pectin pie ee Kit Components 20X Anti phospho EGER Tyr1173 Alexa Fiuor 5S5 Part No CS208204 One vial containing 250 pL 20X AntiEGFR PECy5 Part No CS208205 One vial containing 300 uL 5X Assay Buffer Part No CS202124 One bottle containing 55 mL Fixation Butter Part No C 202122 One bottle containing 13 mL ization Buffer Part No CS202125 One bottle containing 13 mL Permeat Materials Not Supplied Test tubes for sample preparation and storage Tissue culture reagents i e HBSS PBS w o Ca or Mg cell dislodging butfers etc Pipettes with corresponding tips capable of accura
20. utes For smaller cell samples i e 500 000 cells add 500 pL ice cold Permeabiization Buffer 10 Spin down cells at 300 x g for 5 minutes in a tabletop centrifuge and discard supernatant 11 Resuspend calls in 450 pL 1X Assay Buffer per one million cells and aliquot 90 uL per microcentrifuge tube Compatible for the Muse Cell Analyzer Please see Materials Not Supplied section on page 3 12 For single color staining e g adjustment settings add 5 uL of anti EGFR PECyS antibody to the microcentrifuge tube containing the cell suspension 13 For multiplexing add 10 uL of the antibody working cocktail solution as previously described to each microcentrifuge tube containing the cell suspension 14 Incubate call testing samples for 30 minutes in the dark at room temperature 15 Following incubation step add 100 L of 1x Assay Buffer to each microcentrifuge testing sample and centrifuge at 300 x g for 5 minutes on a tabletop centrifuge Discard supematant 16 Resuspend cells in each microcentrifuge tube with 200 pL of 1x Assay Buffer 17 Acquire samples on the Muse Cell Analyzer using the onscreen instructions Setup and Acquisition on the Muse Cell Analyzer Run a System Check prior to performing the assay For information on Muse System Check refer to the Muse Cell Analyzer User s Guide 1 Select EGFR Activation from the main menu EGFR Activation 3 Adjust the instrument settin
21. xa Fluor 555 and 5 ul of anti EGFR PECy5 conjugated antibodies into a centrifuge tube for a final volume of 10 pL total This amount is good for one 1 test Based on the number of tests tubes being performed it is up to the end user to adjust antibody volume amounts at similar ratios e g for 10 tests the working cocktail solution will contain 50 pl of anti phospho EGFA Tyr1173 and 50 ul of ant EGFR for a total of 100 siL Aliquot 10 pL of the working cocktail solution per test tube sample This solution should be prepared as needed but if temporary storage is needed please keep in the dark at 2 8 Assay Instructions Note This assay protocol has been optimized using human A431 cells However this kit is suitable for measuring the extent of EGFR target specific detection of activation via phosphorylation on a variety of human cell types in which EGFR is expressed Alternate species reactivity must be confirmed by the end user 1 Cell Culture and Stimulation Used for example purposes 1 Prepare cells of interest into two tissue culture flasks treated or untreated ovemight in a 37 incubator with 5 CO2 Cells should be at about 90 confluent the next day 2 For the flask labeled Treated treat cells accordingly e g chemically treated using EGF or compound of choice The intent is to induce EGFR activation for the given cell type The other flask labeled untreated will serve as a control 3 Incubate
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