docBIOANKING_Isolation of PBMCs from human blood 14-01-27
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BIOANKING_Isolation of PBMCs from human blood 14-01-27
ISOLATION OF PBMCs FROM HUMAN BLOOD Equipment Centrifuge 1000µL pipete 200µL pipete 100µL pipete 10µL pipete Biosafety cabinet Specification Heraeus Multifuge X3R Monochanel Research Pipette, 1001000µL, variable, manual Monochanel Research Pipette, 1001000µL, variable, manual Monochanel Research Pipette, variable, manual Monochanel Research Pipette, variable, manual NA Catalog No. 75004515 NA Supplier Thermo Fisher Scientific Eppendorf NA Eppendorf NA Eppendorf NA Eppendorf NA Thermo Fisher Scientific Material 50mL tubes 15mL tubes pipetes 5mL Specification Volume up to 50mL, sterile, PP Volume up to 15mL, sterile, PP Serological, pipeting volume 1-7mL Catalog No. 62554502 62547254 4487 pipetes 10mL Serological, pipeting volume 1-13mL 4488 pipetes 25mL Serological, pipeting volume 2-35mL 4250 Plastic Pasteur pipetes Pipete tips Pipete tips TIGE ETROITE ST UNITx500 Filtertips, pipeting volume 100-1000µL, sterile Filtertips, pipeting volume 10µL, sterile Reagent Ficoll Supplier Sarstedt Sarstedt Dominique DUTSCHER Dominique DUTSCHER Dominique DUTSCHER 043251S Catalog No. CMSMSL01-01 Supplier EUROBIO Storage/validity In dark PBS Specification Lymphocytes separation Medium D-PBS, 500mL, no Mg, no Ca 14190-094 GIBCO 4-30°C, 36 months Ethanol for disinfection 70% (v/v) NA internal 15-25°C IMPORTANT: Ficoll, PBS and centrifuge MUST be at room temperature (RT) I. Transfer of blood: 1. Disinfect the tube of blood by wiping it with Anios 2. Centrifuge at 300g, 10' at RT II. Plasma collection: 3. Transfer the plasma (supernatant) into a fresh 15mL Falcon tube 4. Aliquot in 3 aliquots: • 2x 1mL – in 2 1mL 2D tubes • 1x 2mL – in 1x 2mL 2D tube 5. Scan tubes in document of the day “LabExMI-SampleTracking dd-mm-yy” 6. Store at -80°C in dedicated boxes III. Ficoll and Isolation of the lymphocytes: I. Resuspend the pellet in PBS (RT) up to 35mL total and pool it in a 50mL tube. II. Take 50mL tubes prefilled with 15mL of Ficoll and slowly layer 35mL of the diluted blood over the Ficoll being careful not to mix – two layers should be clearly separated. • For other volumes keep the ratio diluted blood:Ficoll around 2.3:1 1 III. Securely close the tube and centrifuge at 800g for 20min at RT, BREAK OFF IV. Use a sterile single use Pasteur pipete to recuperate the mononuclear cells layer (ring) and transfer in a fresh 50 ml tube Optional: you can discard the upper phase before recuperating the ring. V. Add cold PBS up to 50ml VI. Centrifuge at 300g for 8min at 4°C VII. Immediately flick out supernatant. VIII. Flick to resuspend pellet IX. Add 3mL cold PBS over the remaining cell pellet and mix to thourougly resuspend X. Take 10 µl of sample to count cells following the protocol “Cell Counting using Casy” and continue as follows: a) DNA isolation (Only for V1 donor) • Transfer 1x106 cells in a 0.5mL 2D tube • Centrifuge 300g for 8min in the micro centrifuge and aspirate supernatant • Scan tube Scan tubes in document of the day “LabExMISampleTracking dd-mm-yy” • Freeze down at -80°C c) RNA isolation • Transfer 1x106 of cells in a 1.4ml 2D tube • Centrifuge 300g for 8min the micro centrifuge and aspirate supernatant • Add 1mL Trizol and vortex for 1 min • Scan tube Scan tubes in document of the day “LabExMISampleTracking dd-mm-yy” • Freeze at -80°C b) EBV-transformation (Only for V1 donor) Take 1x106 cells and process them according to the protocol “EBVtransformation of B cells”, if they are to be processed the same day. If not, prepare one additional aliquot of PBMCs in a 0.5ml 2D tube and freeze it down following the protocol “Freezing of cells”. d) Cell storage Take the remaining cells and freeze them down in two aliquots (0.5ml 2D tubes) following the protocol “Freezing of cells” For the color of the caps for each type of sample refer to the Annex document “Color-code of caps” 2 COLOR-CODE OF CAPS V1 tube type cap type/color Plasma freezer /-80C 1ml 1ml 2ml screw pink screw blue red cap DNA H freezer /-80C 0,5ml pressure blue RNA freezer /-80C 1,4ml pressure yellow EBV Liquid Nitro 0.5ml screw grey PBMCs Liquid Nitro 0.5ml 0.5ml screw blue screw pink DNA E freezer /-80C 1,4ml pressure green V2 tube type cap type/color 1ml 1ml 2ml 0.5ml 0.5ml screw red screw yellow blue cap screw white screw red 1,4ml pressure white Plasma freezer /-80C PBMCs Liquid Nitro RNA freezer /-80C REF tubes (Thermo) BC83791 BC83734 BC83745 BC83742 374221 tubes 1,4ml tubes 0,5ml tubes 0,5ml screw tubes 1ml screw tubes 2ml 3
