Feb 04, 2020
Version 1
Whole Blood Processing (SepMate) V.1
- 1UCSF
- Human Cell Atlas Method Development Community
Protocol Citation: Yang Sun, David Lee, Jimmie Ye 2020. Whole Blood Processing (SepMate). protocols.io https://dx.doi.org/10.17504/protocols.io.ba87ihzn
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 13, 2020
Last Modified: February 04, 2020
Protocol Integer ID: 31743
Abstract
Purpose: To purify PBMCs from 20 or 50mL whole blood (per donor). SepMate tubes will be used with Lymphoprep to ease processing time and effort.
Materials
MATERIALS
50ml Conical Tubes, Green Cap, 25/BagBio Basic Inc.Catalog #CT788-G.SIZE.1PK
DMSOMerck MilliporeSigma (Sigma-Aldrich)Catalog #D1435
SepMate™-50 (IVD) 100 Tubes
STEMCELL Technologies Inc.Catalog #85450
Lymphoprep™ 250 mL
STEMCELL Technologies Inc.Catalog #7801
FBSInvitrogen - Thermo Fisher
Protein LoBind 1.5mL microcentrifuge tubesEppendorfCatalog #0030108116
EasySep™ BufferSTEMCELL Technologies Inc.Catalog #20144
Falcon™ 15mL Conical Centrifuge TubesFisher ScientificCatalog #14-959-53A
Cryovial 2.0 ml round base internal thread screw cap writing area sterilegreiner bio-oneCatalog #121277
DPBS, no calcium, no magnesiumThermo FisherCatalog #14190136
Mr. Frosty Freezing Container, 2mL tubes, Nalgene Mr. Frosty Freezing Container for 1-2mL cryogenic tubes, PC, clear w/ blue lid, 1/Cs.Thermo FisherCatalog #5100-0001
-SepMate-50 (STEMCELL)
-Lymphoprep (STEMCELL)
-EasySep Buffer (STEMCELL)
-DPBS (Ca/Mg free; Fisher)
-15mL conical tubes
-50mL conical tubes
-1.5mL low bind tubes
-ACK lysis buffer
-Freezing media A (100% FBS)
-Freezing media B (20% DMSO in FBS)
-2.0mL Cryovials
-Freezing container
Safety warnings
Please refer to the Safety Data Sheets (SDS) for health and environmental hazards.
Before start
Lymphoprep should be at Room temperature (~20 °C ) before use. It is important to keep it at Room temperature for the density to remain 1.077g/ml.
Purify 20mL or 50mL whole blood using the following protocol:
50mL whole blood21 steps
To purify PBMCs from 50mL whole blood (per donor).
For each donor, 50mL blood will be collected in 5 BD heparin tubes (green top).
Centrifuge blood samples at1200 x g, Room temperature, 00:10:00 swinging-out rotor and brake off within 06:00:00 of collection.
Carefully transfer 3 mL plasma from each tube in a 15mL tube avoiding the buffy coat, use remaining blood for PBMC isolation.
Note
Note: To minimize PBMC loss during plasma separation, leave the clear layer level at least 1cm above the RBC.
Centrifuge plasma at 16000 x g, 4°C, 00:10:00 in fixed-angle rotor (if a high-speed centrifuge for 15mL tubes is not available, aliquot plasma in multiple 1.5mL tubes and centrifuge at 16000 x g ). Transfer plasma to new tubes avoiding the pellet.
Aliquot 1 mL plasma in multiple 1.5mL low bind tubes, freeze at -20 °C on the day of collection and move plasma samples to-80 °C after 24:00:00 .
PBMC isolation
PBMC isolation
Prepare SepMate tubes with Lymphoprep
Prepare 3 SepMate tubes per donor and add 15 mL Lymphoprep below the insert by pipetting into the center hole.
Note
Take care to minimize any air bubbles below the plastic divider.
Video from SepMate manufacturers (~2min)
Dilute whole blood 1:2 with DPBS (Ca/Mg free).
Note
For 105mL total: 35 mL residual blood and 70 mL DPBS .
Slowly pipette 35 mL diluted blood down the side of the tube.
Note
Some mixing may occur between the medium and blood, but take care not to mix under the divider.
Unlike conventional gradient separation, do not tilt the tube when adding diluted blood.
Spin at 1200 x g, 20°C, 00:15:00 with brakes ON.
Note
Prepare three destination 50mL tubes (~30mL per Sepmate tube will be yielded.)
When the spin finishes, pour off the top layer from the SepMate tubes into your prepared destination tubes.
Note
Do not invert the tubes for more than 2 seconds.
Spin at 450 x g, Room temperature, 00:10:00 with brake ON.
Note
First wash at higher RCF because gradient medium is mixed with the cells.
Pour off supernatant and combine pellets in three tubes to one, add EasySep buffer to 50mL.
Spin at 350 x g, Room temperature, 00:10:00 with brakes ON.
Note
If yield is critical, ensure that the supernatant is clear; opaque supernatant indicates incomplete centrifugation of cells. If necessary, perform the spin at 450 x g .
Pour off supernatant, wash in 50 mL EasySep buffer for a second wash.
Spin 350 x g, Room temperature, 00:10:00 with brakes on.
Pour off supernatant and resuspend in 20 mL EasySep buffer .
Perform cell count.
Spin at 350 x g, 00:10:00 and resuspend in appropriate volume of freezing media A to get 10e7 cells/mL.
Prepare cryovials with 500 µL freezing media B .
Add 500 µL cell suspension from previous step to cryovials and gently mix by pipetting.
Place in freezing container for 1 day at -80 °C and move to LN2 tank for long term storage on the following day.