KuiqpicK User manual
Contents
1. microscope for ventilation 3 Always use the power supply and cord provided by with the unit If a proper power supply and cord are not used product safety performance cannot be warranted 4 When installing KuiqpicK route the power cord away from the microscope frame and KuiqpicK unit 5 Always ensure that the grounding terminal of the KuiqpicK unit and that of the wall outlet are properly connected If the unit is not grounded NeurolnDx cannot warrant electrical safety 6 Never allow metal objects to penetrate any openings on the microscope frame or KuiqpicK unit as this could result in user injury and damage to the instrument 7 Do not insert objects into the capillary holder other than filters and disposable capillary units DCUs 8 When KuiqpicK is not in operation be sure to turn the power off and disconnect the power cord from the connector socket of the KuiqpicK unit or from the wall power outlet 9 Dispose used capillaries DCUs into a biohazard glass waste container according to regulations Caution KuiqpicK components contain hazardous materials KuiqpicK should be disposed according to local regulations 3 Components and Accessories KuiqpicK 1 2 comes equipped with an Olympus CKX41 inverted microscope Olympus America Inc KuiqpicK unit Sampler Head at top with adjustable LED ring light Vacuum Line and Electric Cables Control Box with vacuum module and a power supply wit2. Plug the female cord plug into the power jack located in the rear inside wall of the KuiqpicK side chassis and plug the power cable into the outlet Optional Accessories KuiqpicK is equipped with a trinocular port A camera and or video system can be mounted with an appropriate adapter not supplied Contact your local Olympus sales representative for appropriate adapter and camera options NOTE The power switch and the light intensity adjustment knob on the Olympus CKX41 microscope are nonfunctional on a KuiqpicK 1 2 unit 5 Using KuiqpicK Terminology Starting position Highest vertical position for the DCU the initial position when the unit is first turned on and capillary has been mounted or after it is reset Home position Calibration of Collecting Dissecting position of the DCU tip determined by the user Homing is performed prior to every collection dissection procedure Standby position 1 mm above the Home position DCU will be lifted to Standby position when the Orange Home button is pressed Figure 1 DCU returns to Standby position after each sampling until the Home position or the system has been reset Starting position iil i ds Standby position agi DCU tah Sample m 1mm Side LZA CA Power To turn power on press the Green pushbutton on the side chassis The button will illuminate in depressed position when turned on Press again to turn unit off Figure 1 Filter and DCU attachm
3. Wear gloves and protective eyewear when handling DCUs containing glass capillaries 1 Start KuiqpicK by pressing the Green power button once the button will light up when depressed 2 Lift the KuiqpicK head Figure 2 to attach filter and DCU Caution DCUs have sharp tips Handle with care Improper handling may cause injuries Never contact the capillary tip to avoid injury and contamination 3 To attach the filter and DCU lift the head and attach a filter directly to the male luer connecter on the KuiqpicK sampler head 4 Carefully handle new DCU by its hub base and attach directly onto the filter from entering KuiqpicK s mechanical parts When KuiqpicK is not in use attach a Caution Always use a filter with the DCU to prevent biological material and liquids N new filter unit to prevent foreign objects from entering the system until next use 5 Place a tissue slide on the stage and add buffer to the tissue Bring KuiqpicK head back down 6 Position the slide to a clear spot on the slide adjacent to the tissue sample for calibration 7 Using the White Positioning buttons carefully bring the DCU down until the capillary tip touches the surface of the buffer NOTE Quick button pressing will move the capillary 1 5 um per step Holding down the button for 5 seconds will increase the speed to 350um s Use caution aaa o 11 10 Use the linear stages micromanipulators to locate the DCU halo
4. and position the halo adjacent to the tissue Figure 3A Use coarse and fine focusing knobs to focus on the tissue Slowly bring the DCU down until the annulus of the capillary tip comes into focus as a bright green ring Figure 3B Lower the DCU until the capillary tip comes in contact with the tissue Figure 3C It is helpful to move the microscope slide with the mechanical stage in order to determine if the DCU tip has made contact If the DCU tip has made contact with the tissue proceed to step 11 If the DCU tip has made contact with the slide surface a slight motion of the mechanical stage will result in the simultaneous movement of the capillary tip If the tug of the slide movement is observed press the White Positioning up button several times until the motion of the mechanical stage no longer pulls the capillary tip This assures that the lowest travel of the capillary tip will not break the tip upon downward movement during dissection 11 Using the linear stages align the annulus of the capillary tip to the center of the ocular crosshairs Caution Bringing down the DCU too low will cause the capillary tip to break against the sample slide or plate If you suspect that a tip has been broken be sure to check the surrounding area for broken glass before replacing the DCU to avoid injury or damage Figure 3 Three step procedure for DCU calibration A Initial finding of DCU tip halo shown with red arrow in the field
5. slide surface with aerated ACSF using a dropper or a pipettor and follow instructions in DCU Calibration for Tissue Microdissection and Cell Tissue Collection sections Detach DCU and transfer collected samples as described above to avoid liquid Caution Stop when the capillary shaft is filled to just below the hub with buffer transfer beyond the filter into the system NOTE DCUs with ID 100m diameter and greater are recommended for adult rat brains NOTE For sample dissection video visit our website www neuroindx com or YouTube channel http www youtube com user NDXInc Ejecting Cell Tissue from DCU 9 Prior to removing a DCU from KuiqpicK head pull the syringe plunger back and attach a filter to the syringe tip 10 Carefully remove the DCU from KuiqpicK head and affix to the filter on syringe 11 Carefully position the DCU tip into preloaded buffer and slowly eject tissue into a 1 5mL tube 12 Add 50 uL of 0 05 trypsin 13 Incubate at 37 C for 10 minutes iino 22 14 Add 450 ul of Hank s balanced saline solution HBSS and centrifuge at 1500 rpm for 5 minutes 15 Remove the supernatant 16 Add 800 ul of NB and dissociate cells by aspirating with a pipette 17 Plate the cells and incubate at 37 C in a 5 CO2 atmosphere Media NB 50 ml Neurobasal medium 48 ml Penicillin Streptomycin 100X 500 ul L glutamine 200 mM 2 mM final 500 ul Heparin 5 mg ml 2 ug mL fi
6. 0 in 508mm L x 11 5 in 292 mm min W 15 in 381mm max W 5 Dimensions amp Weight x 16 in 406 5mm H 6 Pump Vacuum range up to 22 Hg Input 24VDC Linear travel step 0 0015 mm fuller aeiater Maximum travel 8 9 mm Input 5 VDC Indoor use 8 Operating environment Altitude max 2000 meters Ambient temperature 5 C to 40 C 41 F to 104 F Maximum relative humidity 80 for temperature up to 31 C 88 F Supply voltage 100VAC to 240 V AC 50 60 Hz 28 9 System Performance Resolution Single Cell Vacuum duration Ts seconds 0 1sto1 0s 4 4 to 22 Hg Vacuum strength Hg Available DCU IDs um From 10 to100 um Acquisition speed Hg Ts seconds Minimum settings 4 4 0 1 sec 1 3 s Maximum settings 22 0 1 0 sec 2 25 Acquisition sample volume Hg Ts DCU ID 4 4 0 1 sec 20 um 10 to 30 nl 22 0 1 0 sec 20 um 1 4 to 2 2 ul 4 4 0 1 sec 30 um 35to55nl 22 0 1 0 sec 30 um 1 5 to 2 8 ul 4 4 0 1 sec 40 um 70 to 100 nl 22 0 1 0 sec 40 um 4 7 to 4 9 ul Cell collection speed cells minute from tissue sections 12 0 1 5 cells min Rat Purkinje cells cerebellum 12 0 1 5 cells min Mouse anterior horn motor neurons Cell collection speed cells minute from adherent cell cultures Up to 25 cells min SH SY5Y human neuroblastoma cell line Up to 25 cells min Chinese hamster ovary cells CHO acquisition vol
7. CU Size DCU size should be selected based on the size of cells of interest and the confluence of cultures DCUs with internal diameter ID greater than the cells are recommended when collecting cells for recultivation and clonal expansion i e when collecting cells of 20um diameter a DCU with ID 30um should be used to maximally maintain cell viability DCUs with lt 30um diameters is recommended when working with smaller cells or cultures with gt 70 confluence However DCUs with smaller ID may cause some damage to cells Collecting Individual Cells To collect cells position the desired cell under the center of crosshairs and press the Black Sample button DCU is lowered and cell is aspirated into the capillary It is recommended that no more than 30 minutes be spent per plate However timeframe for cell collection is dependent on cell type and sensitivity When collecting fluorescently labeled cells Figure 5 calibrate the DCU under bright field as described above then turn off LED ring light Turn on the fluorescent illumination to locate 17 desired labeled cells Turn the LED ring light on and position the cell under the center of the crosshair Turn fluorescent illumination off to prevent bleaching of cells After collecting the cell fluorescent illumination may be turned on again and the process is repeated NOTE Optimal vacuum levels and duration can vary depending on the type of cell cultures and confluence of the cells It is r
8. Ny NEVUROINDX KuiqpicK 1 2 Cell and Tissue Acquisition System User Manual Contents mM A UU N e 10 11 Introduction Safety Components and Accessories Initial Set Up Using KuiqpicK Initial KuiqpicK Training Exercises DCU Calibration for Tissue Microdissection DCU Calibration for Adherent Cell Cultures Cell Tissue Collection DCU Re Calibration Function after Lifting KuiqpicK Head o a OO gog Sample Protocols a Protocol 1 Collection of Individual Adherent Cells from Culture Dishes b Protocol 2 Sucrose Treated Frozen Brain Tissue Microdissection c Protocol 3 Microdissection of Native Brain Tissues d Protocol 4 Microdissection of Microcapillary Cell Walls from Mouse Heart Muscle Tissue Troubleshooting Technical Specifications System Performance Warranty and Liability Contact 16 17 17 19 21 24 26 28 29 30 30 1 Introduction KuiqpicK 1 2 a capillary based cell and tissue acquisition system is the key to reliable precise and affordable cell and tissue sample acquisition prerequisite for a range of in vitro studies including cell and region specific tissue experimentation and single cell analysis This vacuum assisted capillary based system coupled to an inverted microscope allows for rapid and efficient acquisition of specific cells from adherent cell cultures based on their morphology location or fluorescent label It can also collect single cells
9. alibration for Adherent Cell Cultures NOTE Each DCU must be calibrated before sampling For instructional video visit our website www neuroindx com or YouTube channel http www youtube com user NDXInc n Caution Wear gloves and protective eyewear when handling DCUs containing glass capillaries TIP When working with attached cells it is recommended to wash cells with fresh medium to remove dead cells 1 Start KuiqpicK by pressing the Green power button once the button will light up when depressed 2 Lift the KuiqpicK head Figure 2 to attach filter and DCU Caution DCUs have sharp tips Handle with care Improper handling may cause injuries Never contact the capillary tip to avoid injury and contamination 13 3 Attach filter directly to the male luer connecter on the KuiqpicK head 4 Carefully handle a new DCU by its hub base and attach it directly onto the filter and liquids from entering KuiqpicK s mechanical parts When KuiqpicK is not in use Caution Always use a filter with the DCU The filter will prevent biological material attach a new filter unit to prevent foreign objects from entering until next use 5 Place a cell plate dish on the stage and lower the KuiqpicK head 6 Using the White Positioning buttons carefully bring the DCU down until the tip of capillary touches the surface of the liquid Quick button pressing will move the capillary 1 5um per step Holding down the button
10. ay be replaced by 1xPBS e Place on ice Inhibitor Solution a Add 1 proteinase inhibitor tablet in 250ml MEM or 1xPBS Tissue Preparation 1 Anesthetize the animal with 50 mg kg Nembutal or other anesthetic approved by your ARC Flush or perfuse the animal with standard PBS Remove the heart and other organs and sink them in 15 20 Sucrose in PBS at 4 C overnight Flash freeze heart and organs in 2 methylbutane on dry ice If not for immediate use store tissue at 80 C Prepare cryosections from 10 to 25um thickness Stain slides with 0 025 cresyl violet for 10 seconds and gently wash with PBS Apply Dissociation Solution to sections and incubate at room temperature for no more than 30 minutes Tilt the slide onto an absorbent surface e g paper towel and soak away the dissociation solution aaa eee 24 9 Gently wash tissue sections with Inhibitor Solution Tissue Microdissection and Collection 10 Dry the back of the slide and place it on KuiqpicK microscope stage 11 Cover the slide surface with Inhibitor Solution using a dropper or a pipettor and follow instructions in DCU Calibration for Tissue Microdissection and Cell Tissue Collection sections Detach DCU and transfer collected samples as described above to avoid liquid i Caution Stop when the capillary shaft is filled to just below the hub with buffer NOTE transfer beyond the filter into the system Selecting DCU Size DCUs w
11. cell culture cell confluence and tissue type It is recommended that a test sample be used to determine the optimal values of vacuum level and duration prior to sampling Start at the lowest setting for vacuum level and duration and slowly increase them in turn 2 Position the area for acquisition on the cell plate tissue slide to the center of the ocular crosshairs where the DCU has already been positioned Under the microscope the annulus of the capillary tip is where the cell tissue will be collected 3 Push the Black Sample button to acquire the targeted cell tissue 4 Working quickly repeat until desired number of cells tissue has been collected or until DCU has reached its capacity Stop when the capillary shaft is filled just below the hub with buffer 5 Make sure that a syringe with a male luer lock has been prepared with the plunger slightly pulled back and a filter attached to the tip Figure 4 To empty the contents from the DCU lift the KuiqpicK head and carefully detach the DCU Affix the DCU to the filter on the prepared syringe Figure 4 Using both hands hold the DCU in a receptacle e g microcentrifuge tube and eject contents by gently pushing the plunger down Handle the DCU extremely carefully to avoid to break the DCU tip when transfer Caution The fine tip can be fragile when it comes in contact with the tube wall the collected samples Moreover because small amounts of capillary contents can 15 spill
12. during syringe attachment the tip should be held within a tube while attaching the syringe 6 Attach DCU to KuiqpicK head and re calibrate to continue collection e DCU Re Calibration Function after Lifting KuiqpicK Head After setting the Home position for sampling collecting your samples and lifting the head to remove your DCU the instrument will remember your Standby position NOTE Because of the slight variability in the lengths of the DCUs avoid the use of this feature when switching DCUs to prevent accidental breakage of the DCU 1 Attach the same DCU to KuiqpicK and bring the head back down to its upright position 2 Press both White Positioning buttons simultaneously to bring the DCU down to the previous Standby position 3 Calibrate by lowering the DCU until the capillary tip reaches the same level as your tissue cells 4 Press the Orange Home button to set the new Home position which will lift the DCU by 1mm to the new Standby position 5 Press the sample button to collect tissue cell Figure 4 DCU and filter affixed to a luer lock sterile syringe 1 DCU 2 filter 3 syringe 16 6 Sample Protocols a Protocol 1 Collection of Individual Adherent Cells from Culture Dishes Various adherent cell cultures including human neuroblastoma cell line SH SY5Y Chinese hamster ovary CHO cells human melanoma MDA MB 435 cells and various primary cultures such as neural proge
13. ecommended that a test cell culture dish be used to determine the optimal values of vacuum level and duration prior to sampling Start at the lowest settings for vacuum level and duration and slowly increase them in turn Ejecting Cells From DCU Prior to removing a DCU from KuiqpicK head pull the syringe plunger back and attach a filter to the syringe tip Figure 4 Carefully remove the DCU from KuiqpicK head and affix to the filter on syringe Carefully position the DCU tip into preloaded media or buffer and slowly eject cells Reculturing and Clonal Expansion Preconditions Some cell types require preconditioned media same media where the cells were grown in order to grow single cell or a small number of cells If cells do not take after attempting to reculture eject collected cells into filtered preconditioned media in which the cells were originally cultured The culture conditions to grow single cells would be optimized by users which will vary depending on cell types Figure 5 Collecting fluorescently labeled cells A Initial identification of fluorescently labeled cells under fluorescent illumination B Under bright field fluorescent label is still visible Bright field is used to position the cell of interest under the crosshair for collection and to visualize cell collection into the DCU Fluorescent illumination should be turned off to prevent bleaching of label prior to sampling C Upon collecting the cell turn on f
14. ent Lift the KuiqpicK head back to expose the male luer connector Holding the outer rim of the filter connect the female luer lock connector of the filter to the male luer connector on the KuiqpicK head Next attach a DCU to the filter As the KuiqpicK head is brought back down the green horizontal LED will light Figure 2 and a glass capillary component Always handle a DCU by the hub base to avoid Caution DCU consists of two parts the hub base with a female luer lock connector injury and damage to the capillary X Y control To center the tip of the DCU in the field of view use the manual dials Figure 2 on the linear stages at the base of the KuiqpicK head where it mounts to the microscope stage Positioning To position the tip of the DCU along the z axis use the White buttons Figure 1 to move up and down A single quick press will move the DCU by 1 5 um When either of the White buttons is held down for more than 5 seconds the DCU will move at a rate of 350 um s Home Use the White Positioning buttons to set the Home position for sampling i e the desired position of the DCU s tip on the z axis for sampling Figure 1 When the capillary tip comes in contact with the tissue surface press the Orange Homing button Figure 1 When the Home position is designated the Home button will illuminate and DCU will move 1 mm above that position on the z axis to the Standby position Vacuum level and duration cont
15. for 5 seconds will increase the speed to 350um s Use caution Use the linear stages Figure 2 to locate the DCU halo Use coarse and fine focusing knobs to focus on the cells Slowly bring the DCU down until the annulus of the capillary tip comes into focus as a green ring Position the green ring over a clear spot Continue to lower the DCU until the tip has made contact with a cell It is helpful to move the plate dish with the mechanical stage in order to determine if the DCU tip has made contact with a cell or the plate dish surface If the DCU tip is just above the slide surface the edge of the cell will press against the capillary tip See figure below If contact is made proceed to step 10 9 Ifthe DCU tip is in contact with the slide surface a slight motion of the mechanical stage will result in the simultaneous movement of the capillary tip If the tug of the slide movement is observed press the up button several times until the motion of the mechanical stage no longer pulls the capillary tip This assures that the lowest travel of the capillary tip will not break the tip upon downward movement during dissection 10 Using the linear stages align the annulus to the center of the ocular crosshairs Figure 2 Caution Bringing down the DCU too low will cause the capillary tip to break against the sample slide or plate If you suspect that the tip has been broken be sure to check the surrounding area for broken glass before replacin
16. from suspension cell cultures and individual multicellular spheres from three dimensional 3D cell cultures KuiqpicK collects cells without compromising cell viability thus enabling primary culturing or recultivation of the collected single cells In addition to the collection of individual cells from culture dishes KuiqpicK performs isolation of single cells and microdissection of subanatomical regions from various brain tissue samples prepared by different methods such as fresh frozen tissue sucrose treated tissue and fresh live tissues with minimal contamination of surrounding components while leaving the intracellular structure and molecules intact Moreover it works with thicker tissue sections up to 500 um suitable for an analysis that requires large amounts of sample material such as proteomics Samples collected using KuiqpicK may be used for a wide range of downstream applications and techniques used in modern molecular biology targeting both protein and nucleic acids including but not limited to quantitative RT PCR global gene expression epigenetic and proteomics studies 2 Safety 1 Handle glass capillaries with care Capillaries have sharp points and can break on or under skin surface if accidently contacted therefore it is strongly recommended to always wear goggles and gloves during handling 2 Install KuiqpicK microscope on a sturdy level surface and allow at least 5 inches of space all around KuiqpicK
17. g the DCU to avoid injury or damage 11 Press the Orange Home button the button will light up when calibrated to set the calibrated Home position The DCU will remain on Standby position 1 mm above the sampling position 12 To re calibrate simply press both White buttons simultaneously to bring the DCU back to Home position Re position the capillary tip using the White Positioning buttons and then push the Orange button to set the new Home position To cancel re calibration press both White buttons simultaneously instead of pressing the Home button This will bring the DCU back up to the previous Standby position aan o 14 13 You may re calibrate at any time during the sampling procedure to designate a new Home position 14 Proceed to Cell Tissue Collection Section NOTE When the KuiqpicK head is lifted the green horizontal light will turn off and the DCU will reset and move up to its initial starting position The DCU must then go through the calibration process again Caution When the KuiqpicK head is lifted the DCU will return to its initial starting position Do not try to detach the DCU immediately after lifting the KuiqpicK head Wait until the DCU stops moving completely to avoid causing damage to the KuiqpicK sampler unit d Cell Tissue Collection 1 Adjust values for the white LED ring light vacuum pressure and duration NOTE Optimal vacuum levels and duration can vary depending on the type of
18. h cord Ready to use Disposable Capillary Units DCUs attachments for cell collection and brain tissue dissection and filter attachments are sold separately NOTE The camera camera adapter and fluorescence illumination system as shown in the figure below are not included and must be purchased separately Inverted microscope with mechanical stage XY stage mount KuiqpicK Sampler Head KuiqpicK Vacuum Line and Electric Cables KuiqpicK Control Box Light adjustment dial vacuum dials and DCU controls Se ee ae 4 Initial Set Up All necessary components are included in the packing Make sure that the following items have been received upon opening the package KuiqpicK and microscope Olympus CKX41 body Mechanical XY stage x1 Trinocular observation head x1 Eyepiece x2 Crosshair reticle with reticle holder x1 Objectives one of each 4X 10X 20X Hex wrench x1 Stage plate round silver oS ee eo eo Stage plates black set of 3 10 Power supply and cord for KuiqpicK one of each Proper installation of all parts is required for KuiqpicK to work properly Make sure the following steps are completed before attempting to use the system Refer to the Olympus CKX41 manual for operation and installation specific to the microscope Objectives Lower the revolving nosepiece using the coarse adjustment knob towards the back Remove the dust prevention cap Screw the objectives starting with the
19. ices herein are the trademarks service marks registered trademarks or registered service marks of their respective owners NeurolnDx Inc has made every effort to ensure that this manual is as complete and as accurate as possible but no warranty or fitness is implied The information provided is on an as is basis NeurolnDx Inc shall have neither liability nor responsibility to any person or entity with respect to any loss or damages arising from the information contained in this manual 30
20. ith ID lt 30um for single cell collection ID 20 100um for subanatomical regions and ID 60 80um for microcapillaries are recommended NOTE For sample dissection video visit our website www neuroindx com or YouTube channel http www youtube com user NDXInc Ejecting Cell Tissue from DCU 12 13 14 15 16 Prior to removing a DCU from KuiqpicK head pull the syringe plunger back and attach a filter to the syringe tip Carefully remove the DCU from KuiqpicK head and affix to the filter on syringe Carefully place the DCU tip into a microcentrifuge tube preloaded with buffer and slowly eject cell tissue If there is remaining tissue inside the capillary shaft carefully and slowly load the attached syringe with Inhibitor Solution to rinse the DCU and then release the contents into the microcentrifuge tube Spin cells at 500 x g for 5 minutes and remove the supernatant to remove the Inhibitor Solution Samples may then be used immediately or frozen on dry ice and stored at 80 C for later use References 1 Alisina Janivette Leach Steven D and Baily Jennifer M 2013 The incorporation of polaxamers during pancreatic tissue dissociation allows isolation of high quality RNA from FACS sorted pancreatic cells The Pancreapedia Exocrine Pancreas Knowledge Base DOI 10 3998 panc 2013 10 25 7 Troubleshooting If problems with KuiqpicK occur by factors other than manufacturing defects please re
21. lowest magnification into the revolving nosepiece from the left side of the microscope Turn the nosepiece clockwise and mount the remaining objectives in ascending magnification order Objectives should be cleaned periodically Cover unused threaded position with the dust prevention cap to prevent foreign objects from entering Observation Head 1 Loosen the head locking screw on the observation head and remove the dust cover caps on the dovetail cavity and the observation head 4 2 Mount the observation head by engaging the dovetail at the bottom of the head into the cavity in the microscope s arm 3 Tighten the head locking screw after positioning the observation head as desired Eyepieces 1 Remove the protective caps from the observation tubes 2 Insert the reticle in one of the 10x eyepieces and secure it by screwing in the reticle holder 3 Insert the 10x eyepieces into the ocular sleeves Interpupillary Distance IPD and Diopter 1 Looking through both eyepieces move the left diopter until both left and right fields of view perfectly coincide This is your proper IPD 2 Looking through the right eyepiece only use the coarse and fine focus adjustment knobs to bring the specimen into focus Adjust the setting to 0 on the left eyepiece and look through with the left eye Bring the specimen into focus using only the diopter setting Power supply and cord Connect the 24V power supply and the cord
22. luorescent illumination to resume collection NOTE Change the DCU and filter after each collection procedure DCUs may be reused by washing with a syringe and sterilizing with ethanol DCUs can also be autoclaved However NeurolnDx recommends using a new DCU for each collection procedure to avoid contamination aan 18 b Protocol 2 Sucrose Treated Frozen Brain Tissue Microdissection This protocol describes the isolation of single cells cell clusters and subanatomical regions from sucrose treated brain slices Sucrose treated brain tissue keeps brain morphology intact and thus ideal for microdissection Furthermore this optimized protocol yields high quality RNA from the microdissected material Materials e Surgical instruments e Standard animal perfusion apparatus and setup e Cryostat e Standard Phosphate Buffered Saline PBS e Sucrose 15 20 in PBS filtered e 2 methylbutane e Dry ice e Glass microscope slides e 0 025 Cresyl violet 0 01 toluidine blue hematoxylin or any vital dyes e Pipettor and sterile pipette tips e KuiqpicK disposable capillary units DCU with appropriate internal diameter ID for the application ID lt 30 um single cell collection ID 20 100 um for subanatomical regions Recommended tissue preparation Flush animal with standard preperfusion PBS phosphate buffer saline Remove the brain and sink in 15 20 Sucrose in PBS at 4 C overnight Flash freeze the brain in 2 methylbuta
23. nal 20 ul B 27 50X 1 ml FGF2 25 ng ul 20 ng ml final 40 ul EGF 100 ng ul 10 ng ml final Sul d Protocol 4 Microdissection of Microcapillary Cell Walls from Mouse Heart Muscle Tissue This protocol is suitable for dissecting most difficult to dissect tissue and isolating high quality RNA from collected samples Materials Liberase DL Research Grade Roche REF 05 401 160 001 5mg vial Proteinase inhibitor Sigma tablets SigmaFast 8820 Kolliphor P188 Sigma 15759 also known as Pluronic F 68 1M sterile CaCl MEM without L glutamine FBS or antibiotics Corning 1x Phosphate buffer saline PBS Cryostat Ice 23 Reagents Prepare prior to the day of microdissection 1 P188 stock solution 15mM 100x stock solution place in a 37 C water bath to dissolve filter sterilize and store at 4 C in the dark 2 Liberase DL Stock Solution a Slowly thaw enzyme Liberase DL lyophilized form on ice for 30 min b Add 2ml of MEM alternatively 1x PBS may also be used c Keep on ice resuspend by pipetting Do not vortex Stock may be kept at 4 C for up to a month Alternatively aliquot into smaller volumes 200 ul and store at 20 C Use or discard an aliquot after thawing Do not re freeze Prepare on the day of dissection 1 Dissociation Solution mix the following components to make 10ml of solution a 200ul Liberaze stock solution b 75ul 1M CaCl c 100 ul P188 d MEM to 10ml MEM m
24. ne on dry ice If not for immediate use store tissue at 80 C Prepare cryosections from 10 to 100 um thickness For single cell collection with KuiqpicK cut sections at lt 30 um Stain slides with 0 01 toluidine blue or 0 025 cresyl violet for 10 seconds Wash with standard PBS Also see Application Notes on our website http www neuroindx com KuigpicK application notes Caution Dyes should be used at lower than standard concentrations Overstaining will cause stiffening of tissue samples thus making them difficult to microdissect Microdissection or Collection of Single Cell from Tissue Slices Dry the back of the slide after PBS wash and place the slide on KuiqpicK stage It is critical to keep the tissue section moist at all times by adding either PBS or 15 sucrose in PBS during longer sessions of microdissection High quality RNA may be isolated from brain tissue samples 19 collected within 1 hour Attach a DCU to KuiqpicK head Follow instructions in DCU Calibration for Tissue Microdissection and Cell Tissue Collection sections NOTE Optimal vacuum levels and duration can vary depending on the type of samples being dissected It is recommended that a test slide be used to determine the optimal values of vacuum level and duration prior to sampling Start at the lowest settings for vacuum level and duration and slowly increase them in turn Selecting DCU Size DCUs of different diameters should be used based on the
25. nitors skin fibroblasts etc can be used for the collection of individual cells using KuiqpicK The collected cells are viable and may be used for recultivation and clonal expansion as well as for single cell analysis including protein and nucleic acid analyses See Application Notes on our website http www neuroindx com KuiqpicK application notes NOTE Recommended medium for neuroblastoma cell line SH SY5Y Chinese hamster ovary CHO cells and human melanoma MDA MB 435 cells ATCC Manassas VA Dulbecco s Modified Eagle Medium Nutrient Mixture F 12 DMEM F 12 Life Technologies Grand Island NY supplemented with 10 v v heat inactivated fetal calf serum containing 2mM glutamine and 1 antibiotics penicillin streptomycin Cells should be maintained at 37 C in a humidified 5 CO2 atmosphere For some tightly adherent cells we recommend non enzymatic Cell Dissociation Solution e g Sigma C5789 Cell collection with KuiqpicK from cultures It is recommended for cell cultures to not exceed 50 confluence for single cell collection although collections may be performed at higher confluence Prior to cell collection media should be removed from the plate dish and cells should be gently washed once with fresh pre warmed medium to remove dead floating cells Replenish media and place plate dish on microscope stage Proceed with cell collection as described in Calibration for Cell Cultures and Cell Collection sections Selecting D
26. ntaining 1 v v Penicillin Streptomycin 2uM final concentration L glutamine 0 04 v v Heparin 2 v v B 27 20ng ml fibroblast growth factor FGF2 and 10ng ml epidermal growth factor EGF e Ice NOTE ACSF may be prepared the night before and cooled to 4 C in a refrigerator Tissue Preparation Specimen preparation may be performed according to standard protocols Described below is the preparation used for KuiqpicK protocol 1 Practical Electrophysiological Methods A Guide for In Vitro Studies in Vertebrate Neurobiology 1992 Eds Helmut Kettenmann and Rosemarie Grantyn Wiley Liss New York 21 1 Aerate ACSF with carbogen 10 20 minute before the experiment 2 Anesthetize the animal with 50 mg kg Nembutal or other anesthetic approved by your ARC 3 Open the chest and flush the animal through the aorta with cold aerated ACSF for 3 5 minutes 4 Excise the brain rinse with cold ACSF and remove excess liquid with sterile napkin 5 Keep brain in buffer consisting of Krebs Ringer solution saturated with carbogen 95 O2 5 CO2 on ice until mounted onto glass slides 6 Glue the brain on the vibratome platform with fast glue e g medical device adhesive Loctide 4014 7 Slice 200 300um sections and carefully place a tissue slice onto a glass slide and keep moist with ACSF Keep slides on ice Tissue Microdissection and Collection 8 Dry the back of the slide and place it on KuiqpicK stage Cover the
27. of vision B Lowering the DCU until the DCU tip and tissue section are both in focus C Moving the XY mechanical stage to test if the DCU tip is in contact with the tissue section surface 12 Press the Orange Home button the button will light up when calibrated to set the calibrated Home position The DCU will remain at the Standby position 1 mm above the Home position i e sampling position 13 To re calibrate simply press both White buttons simultaneously to bring the DCU back to Home position 12 Re position the capillary tip using the White Positioning buttons and then push the Orange button to set the new Home position To cancel re calibration press both White buttons simultaneously instead of pressing the Home button This will bring the DCU back up to the previous Standby position 14 You may re calibrate at any time during the sampling procedure to designate a new Home position 15 Proceed to Cell Tissue Collection Section NOTE When the KuiqpicK Head is lifted the green horizontal light will turn off and the DCU will reset by moving up to its initial starting position The DCU must then go through the calibration process again Caution When the KuiqpicK head is lifted the DCU will return to its initial starting position Do not try to detach the DCU immediately after lifting the KuiqpicK head Wait until the DCU stops moving completely to avoid causing damage to the KuiqpicK sampler unit c DCU C
28. rol The two dials at the front of the side chassis Figure 1 control the vacuum level from 2 Hg 1 to maximum of 22 Hg 10 and the vacuum duration from 100 ms 1 to maximum of 1 s 10 Depending on the sample and the desired result various vacuum level and duration may be used We recommend testing these parameters prior to any collection dissection Sample After the Home position has been set pressing the Black Sample button Figure 1 will initiate sample collection by bringing the DCU down to the Home position and activating the vacuum at the selected strength and duration Figure 1 KuiqpicK 1 2 controls include 1 Power on off Green button 2 Positioning White buttons to bring the capillary up or down towards the stage 3 Home Orange button to calibrate capillary DCU for cell and tissue collection and set it in standby position 4 Light intensity top dial 5 Vacuum middle dial increasing vacuum pressure from 1 minimum 2 inches of mercury to 10 maximum 22 inches of mercury 6 Duration bottom dial increasing vacuum duration in increments of 100 milliseconds from 1 shortest 100 milliseconds to 10 longest 1 second 7 Sample Black button for cell tissue collection Figu re 2 KuiqpicK head 1 in its half lifted position for disposable capillary DCU attachment removal shown here with filter 2 and DCU 3 attached In the lifted position the green horizon
29. size of the cells and subanatomical regions of interest ID lt 30um for single cell collection and 1ID 20 100um for subanatomical regions is recommended Figu re 6 Unilateral microdissection of the corpus callosum 1 paratenial thalamic nucleus 2 nucleus reunions 3 and hypothalamic nucleus 4 Tissue was stained with toluidine blue Tissue thickness 50 um Scale bar 250um NOTE Check the DCU carefully during dissection to make sure that the samples are being collected into the capillary If not repeat calibration or adjust vacuum level and duration Detach DCU and transfer collected samples as described above to avoid liquid Caution Stop when the capillary shaft is filled to just below the hub with buffer transfer beyond the filter into the system Ejecting Cell Tissue from DCU Prior to removing a DCU from KuiqpicK head pull the syringe plunger back and attach a filter to the syringe tip Carefully remove the DCU from KuiqpicK head and affix to the filter on syringe Carefully position the DCU tip into preloaded buffer and slowly eject cell tissue Cells tissue may be ejected into a microcentrifuge tube containing the desired buffer for subsequent application To remove remaining tissue inside the capillary shaft carefully and slowly load the attached syringe with PBS to rinse the DCU and then release the contents into the microcentrifuge tube If not for immediate use samples may be frozen on dry ice and then s
30. slide The tissue section has been fixed before sectioning Make sure that the tissue is not fixed Tissue sections are thick Mount tissue sections with thickness of 50 um or less 26 6 Orange Home button does not light up White Positioning buttons were simultaneously pressed repeatedly or Homing button was pressed without bringing down the DCU at least 1mm Reset DCU to starting position by lifting up KuiqpicK head or powering off on 7 Orange Home button blinks continuously There is a leak in the vacuum line Make sure that the blue tubing behind the control box is secure The movement of the DCU in the sampler unit is obstructed Check to see if there is anything obstructing the movement of the DCU and remove it 27 8 Technical Specifications Specification Description 1 Illumination Light source 144 LEDs ring light Input 24VDC 2 Focusing mechanism Stage height adjustment mechanism Fine adjustment scale 0 002 per graduation Fine adjustment stroke 0 2mm per turn Total stroke 28mm Co axial coarse and fine focusing on ball drive Coarse adjustment travel per rotation 3 Observation tube 4 Stage Field number 22 Tube tilting angle 30 Interpupillary distance adjustment range 52 75 Size 240 x 160 mm with mechanical X Y stage Movement range 114 x 41 3mm Specimen holder Slide and Petri dish 20
31. tal LED light 7 is automatically turned off It is automatically turned on when the KuiqpicK head is in its upright position The ring LED lights 4 stay on ina fully lifted position The x y position of the DCU is controlled by the knobs 5 on the linear stages 6 The knobs on the right and left sides of the bottom stage control the side to side movement and those on the front and back of the stage control the front to back movement of the DCU to center it above the mechanical XY stage a Initial KuiqpicK Training Exercises For instructional videos visit our website www neuroindx com or YouTube channel http www youtube com NDXInc 1 Start KuiqpicK by pressing the Green power button once the button will light up when depressed Lift Head to attach filter and DCU as described in Calibration section i Caution Wear gloves and protective eyewear when handling DCUs containing glass capillaries 2 Place a marking on a histological glass slide and place it on the mechanical stage with 2 drops of distilled water 3 Using the White Positioning buttons carefully bring the DCU down until the capillary tip touches the surface of the liquid NOTE Quick button pressing will move the capillary 1 5 um per step Holding down the button for 5 seconds will increase the speed to 350 um s 4 When in focus the annulus of the capillary tip appears as a bright green ring 5 Use the knobs on the linear stages Figure 2 to loca
32. te the green annulus ring 6 Locate the marking on slide Using coarse and fine focus adjustment knobs to focus on marking 7 Carefully bring the DCU down until the annulus of the capillary tip comes into focus as a bright green ring 8 Stop lowering the DCU when the capillary tip moves with the glass slide when using the mechanical stage It is helpful to move the slide plate using the mechanical stage to determine if the DCU tip has made contact with the slide plate surface When the DCU tip is in contact with the surface a slight motion of the mechanical stage will result in a movement of the capillary tip 9 Lift the Head to reset KuiqpicK 10 Bring the head back down and repeat steps 1 through 7 10 b DCU Calibration for Tissue Microdissection Each DCU must be calibrated before sampling For instructional video visit our website www neuroindx com or our YouTube channel http www youtube com NDXInc NOTE Depending on the region of interest DCUs of different internal diameters IDs should be used The size of desired cells and cell clusters shall determine the appropriate diameters of the DCUs An average interneuron is about 15 25um For microdissection of brain anatomical regions e g mouse hippocampus 50um ID DCU is recommended Subanatomical regions such as dentate gyrus or cortical layers may be collected with 30um ID DCUs If fine microdissection is required DCUs with lt 30um diameters should be used
33. tored at 80 C 20 NOTE Change the DCU and filter after each collection procedure DCUs may be reused by washing with a syringe and sterilizing with ethanol DCUs can also be autoclaved However NeurolnDx recommends using a new DCU for each collection procedure to avoid contamination c Protocol 3 Microdissection of Native Brain Tissues KuiqpicK is able to dissect live tissues for primary culture and various downstream molecular analyses The following is a sample protocol for the collection of live cells from brain tissue to be used for culturing KuiqpicK can be applied to dissect live cells from other tissues such as tumors for primary culture using suitable protocols Under appropriate extracellular and sterile conditions ensuring cell viability fresh adult brain tissue containing live cells may be used for dissection for up to 8 hour See Application Notes on our website http www neuroindx com KuigpicK application notes Materials e Vibratome e Thermostat bath e Surgical instruments e Artificial Cerebrospinal Fluid ACSF 300 500mlI containing in mM 126 NaCl 1 25 NaH 2PQ a 26 NaHCOs 2 5 KCI 2 CaCl2 2 MgCl2 and 10 D glucose pH 7 4 carbogen 95 O2 5 CO2 ACSF may be replaced by tissue culture medium balanced to carbogen e Dissociation solution 1mM EDTA 0 05 Trypsin e Hanks Balanced Salt Solution HBSS e Neurobasal media with supplements and growth factors Neurobasal media NB see below co
34. ume depends on the DCU ID and sample viscosity 29 10 Warranty and Liability NeurolnDx Inc warrants its products to be free of defects in materials and workmanship for a period of one 1 year from date of purchase unless otherwise specified at time of purchase The foregoing warranty of NeurolnDx Inc shall be of no force and effect if buyer has modified or damaged the product No other warranties of any kind expressed or implied are provided by NeurolnDx Inc NeurolnDx Inc shall have no liability for direct indirect consequential or incidental damages arising from the use results of use or inability to use its products 11 Contact NeuroInDx Inc 1655 E 28 Street Signal Hill CA 90755 Tel 562 424 4611 Fax 562 424 4652 info neuroindx com www neuroindx com Copyright 2015 NeurolnDx Inc All rights reserved No part of this publication may be reproduced stored in a retrieval system or transmitted by any means electronic mechanical photocopying recording or otherwise without written permission from NeurolnDx Inc This manual is sold as part of an order and subject to the condition that it shall not by way of trade or otherwise be lent resold hired out or otherwise circulated without the prior consent of NeurolnDx Inc in any form of binding or cover other than that in which it is published KuiqpicK and DCUs are covered by US and foreign patents and pending patent applications All products and serv
35. view the following guide If you encounter any other problems please contact technical support Problem Cause Solution 1 Tissue or cell is not being picked up into the DCU Vacuum level and or duration are is not high long enough Increase vacuum pressure and or duration Use a DCU with larger tip diameter DCU tip is clogged Maintain humidity between tissue and the DCU tip Change to a new DCU DCU tip is not touching the sample slide Re calibrate the DCU DCU tip has been damaged Change to a new DCU 2 Too much background light for capturing microscope images 3 DCU does not move by pushing the White Positioning buttons Green horizontal LED light on KuiqpicK head Block off light source with masking tape or other opaque object Reflection of light off of the DCU s glass capillary shaft DCU is too short and has reached its lowest position Bring DCU up with White Up button Restart KuiqpicK by powering off and on DCU may have been broken Change to a new DCU 4 Too much liquid is accumulated in the capillary shaft or filter gets clogged Vacuum level and or duration are set too high Decrease vacuum level and or duration Monitor the capillary shaft during collection Stop collection when it is filled to just below the hub with buffer Change the filter attachment 5 Bubbles appear on the surface of tissue
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