STAINING PROTOCOL FOR CYTOMETRY (Réf LabEx #1)

Transcription

1 STAINING PROTOCOL FOR CYTOMETRY (Réf LabEx #1) Aim To provide detailed description of all steps, material and methods used for fluorescent surface staining and preparation of whole blood cells for analysis by flow cytometry. Each «panel» represents a combination of commercially available antibodies against different cell surface markers used to identify descrete subpoplations of immune blood cells. The subpopulations that can be detected using these panels are the following: T cells (naïve, central memory, effector memory, EMRA + ), NK cells, monocytes, dendritic cells, neutrophils, basophils and eosinophils. Materials 1) BIOLOGICAL SAMPLES - Fresh human whole blood, collected, transported and received according to the standardized procedures (See Protocol #2 - Reception and processing of blood). 2) ROOM AND EQUIPMENT The experiments were performed in a dedicated. - Biosafety cabinet: Herasafe KS18 GS (TÜV Nord) - 2 Flow cytometers : MacsQuant Analyzer 10, Miltenyi - Pipetes : µL, µL, µL, 1-10µL & µL (Research, Eppendorf ) - Vortex Genie2 (Scientifique Industrie) - Centrifuges: Megafuge 40R (ThermoFisher) and Megafuge 40 (ThermoFisher) - Liquid handling station Freedom Evo 150 (Tecan) ) PLASTICWARE - BD 5ml polystyrene tubes, Cat.No deepwell plates 2ml, Cat.No , 04282, or Tips : ART10 ( ), ART100 (2065 E- 05), ART200 ( ) and ART1000 (2079) - Disposable 200µL and 1000µL Tips (TECAN) 4) SOLUTIONS - PBS, no Ca no Mg (Gibco) - Red Blood Lysis solution: BD FACS Lysing solution; BD Cat.No (10x) diluted with milliq water 5) ABBREVIATIONS Ab antibody SOP standard operating procedure RT room temperature : 20 to 22 C RBC - red blood cell SOP - Standard Operating Procedure N - number of sample MQ-1/2 MACSQuant 1/2

2 Staining Protocol Prior to sample reception, prepare: 1) Antibodies / premixes Prepare premix of antibodies for each panel calculating the quantities according to the number of samples, calibration controls (+1) that will be received and analyzed that day: - Label 1.5ml tube with the name of corresponding panel - Prepare each premix according to dilutions required for each antibody - Store premixes at +4 C or on ice, protected from light, until use 2) Plasticware - Empty deepwell plates, named MQ1- date and MQ 2- date - For washing the blood: Labelled 5ml tubes with ID or barcode that will be used for each sample PBS (room temperature) ) Reagents: - 20ml per samples of 1x RBC Lysing solution: Dilute FACS lysing solution at 1/10 in water - Prepare 1x Viability dye solution for N x 5 Dilute at 1/1000 dilution in sterile PBS ; 5mL/ donor Keep Viability dye solution at 4 C or on ice, protected from light. Keep RBC lysing solution at room temperature for at least 0min before use. 4) Cytometer: - Turn on cytometers, log on user session and select acquisition mode according to the SOP Wash blood prior to the staining : - Take 2ml of fresh whole blood and add it to earlier prepared 5ml tubes containing 2ml PBS at RT - Centrifuge at 500g for 5min - Discard supernatant - Add PBS (RT) up to 2ml MANUAL STAINING Note: Incubations can be performed under the hood or on the bench at RT without shaking. 1) Prepare 1 x 5ml FACS tube per panel and per donor or 2 deepwell plates for up 12 donors 2) Mix, by pipeting, all Ab premixes prior to using them ) Spin shortly (about 20s) all premixes 4) Add each premix in the corresponding tube or well 5) Transfer 100µl of whole blood in corresponding 5ml tube/well 6) Mix shortly by vortex (tubes) or plate-shaker 7) Incubate 20min in dark at RT For panels including dead cell staining: follow steps 8-11 For panels without dead cell staining: continue by step 12 8) Add 1ml 1x viability dye solution

3 12) 1) 14) 15) 16) 9) Incubate 0 min in dark at 4 C 10) Add 1 ml cold PBS (4 C) 11) Centrifuge 5 min at 500g and aspirate supernatant Resuspend in 2000µl 1x RBC lysing solution and vortex shortly Incubate 15 min at RT protected from light Centrifuge 5 min at 500g, aspirate supernatant Resuspend in 240µl PBS Acquire immediately Semi-automated staining procedure : This protocol should be followed with the provided script for TECAN operations (see Protocol #). The user is advised to follow the dialog boxes provided. Incubations are performed in the automat using a Torrey Pines incubator. Mix, and centrifuge all Ab premixes prior to distributing them into the liquid handler Check liquid waste and tip waste prior to initiating operations; empty if necessary Evo150 worktable layout : 1 = Carrier trough (100ml) positions (Lysis buffer, Viability dye, PBS) 2 = Incubator TorreyPines EchoTherm 2 (Incubations at RT) = Incubator TorreyPines EchoTherm 1 (Incubations 4 C) 4 = Shaker 5 = Tips Rack positions (200µl and 1000µl) 6 = Rack strip 16 position (for 1mm tubes) 7 = Tips waste 8 = Wash station 9 = Arm LiHa (8 channels)

4 Prepare the pipetting robot (requires 15-0min before starting the experiment): 1) Turn on robotic system and launch software a. In the front of automat, click button ON b. On the desktop, double click EVOware icon Startup window appears c. Choose run an existing script and click start your selection d. Choose Staining Script and click run button (green triangle) Runtime controller window appears e. Verify that the option run full script is checked f. Launch script by clicking on run button Automat initializes A pop-up windows appears to define the number of samples (variable) g. Indicate in the appropriate N of sample 2) Prepare the worktable following dialog boxes and validate a. Switch on the Torrey Pines incubators i. The double incubator position on the left should be set to 26 C ii. The simple incubator position on the right should be set to 4 C b. Place the 2 deep-well plates on the left Torrey Pines station i. MQ1 plate on the back ii. MQ2 in the front c. Verify that the 1.5ml rack (aluminum) is at 4 C A dialog box will appears, indicating a standby step, waiting to start the script when samples and premixes are ready. If the premixes are ready and the blood samples are at the washing step, the script can be launched d. Place the premixes in the aluminum rack e. Click OK to start the staining protocol The robot distributes premixes: - premixes T, ILC, Th, B & Lineage for MQ1 plate - premixes PMN, NK, TReg & DC for MQ2 plate At the end of premix distribution, verify correct distributed A dialog box waiting blood samples appears, click OK to continue ) Continue staining protocol with blood samples a. Blood tubes are placed in the 5ml tube rack, first tube in position 1 b. Verify that the barcodes are readable A window appears to set identification of sample: a. Identify samples, one by one With scaner read barcode, one after another; or With keyboard: write ID of each sample Following dialog boxes, the robot places 5ml tube rack (with blood samples) on the rail 2 b. Click OK to continue the staining The robot distributes blood 4) Shake plates when dialog box appears 5) Prepare lysis solution in 100ml container named Lysis 6) Prepare viability solution in 100ml container named Viability

5 Timer starts: 20min of incubation for staining Pop-up windows show time remaining When the staining is complete, a dialog box indicates to place viability solution and lysis solution into the robot. 7) Click OK to continue Automat distributes Lysis in MQ1 A dialog box appears: place MQ1 on shaker and shake MQ1 for 0s Timer starts: 20min of incubation for lysis 8) Click OK to continue The robot distributes Viability solution in MQ2 A dialog box appears to place MQ2 on 4 C incubator Timer starts: 0min of incubation for viability staining 9) Click OK to continue During incubation, the automat mixes by pipeting the wells of MQ1 and adds 1ml more of lysis solution Dialog box indicates to user to centrifuge MQ1 10) Centrifuge MQ1 plate at 500g for 5min 11) Replace on back incubator and click OK Automat aspirates supernatant and distributes 240µl PBS in each well. The user is indicated to transfer MQ1 plate to MQ1 cytometer 12) Click OK to continue with MQ2 plate After completion of viability incubation, dialog box ask to replace MQ2 on left incubator. 1) Replace MQ2 on left Incubator in the front 14) Click OK to continue Automat distributes PBS up to 2ml 15) Centrifuge MQ2 plate at the end of distribution at 500g for 5min 16) Replace MQ2 plate and click OK, automat aspirates supernatant 17) Shake MQ2 plate when indicated, re-introduce the plate and click OK Automat distributes lysis solution 18) Shake MQ2 when the dialog box appears Automat mixes by pipetting and distributes lysis solution up to 2ml Dialog box appears to show the time remaining A dialog box requests user to centrifuge the plate 19) Centrifuge MQ2 plate at 500g for 5min 20) Replace on back incubator and click OK Automat aspirates supernatant and distributes 240µl PBS in each well. The user is indicated to transfer MQ2 plate to MQ2 cytometer