Version: 1.0
A PDF version of this protocol is available for download here.
Isolating and Purifying PBMCs from Whole Blood
For processing whole blood at Parse Biosciences, we recommend:
- EasySep™ Direct Human PBMC Isolation Kit, Stemcell Technologies, Catalog # 19654 EasySep™ Direct Human PBMC Isolation Kit
- We use this with the magnet “The Big Easy” also from Stemcell Technologies, catalog #18001 “The Big Easy" EasySep™ Magnet
The SOP that Parse Biosciences recommends:
- EasySep™ Direct Human PBMC Isolation from Whole Blood.
- From the EasySepTM Direct Human PBMC Isolation from Whole Blood SOP on page 2, “The Big Easy” column (seen below for reference)
Please note steps that are not explicitly in the protocol:
1. Make buffer media with final concentrations of: 1X PBS containing 2% FBS and 1 mM EDTA
2. Add 2 mL of whole blood to the specified tube (14 mL (17 x 95 mm) polystyrene round-bottom tube (e.g., Catalog #38008))
3. Add EDTA to the sample to a concentration of 6 mM EDTA
4. *Follow EasySep™ protocol steps 3-13 as written.* If you started with 2 mL of blood, then there should be ~ 4 mL of purified enriched cell solution.
5. Spin enriched solution 200xg for 10 mins at 4°C.
6. Resuspend in 100 μL Cell Prefixation Master Mix and dilute 1:2 to count cells
7. Move up to 1 million cells into the Cell Fixation process and top up to the correct volume of Cell Prefixation Master Mix using the current version of the Cell Fixation User Guide.
Critical! For processing PBMCs from whole blood, samples may be processed up to 48 hours post-draw. At the time of the blood draw, it is strongly recommended to immediately place and retain samples at 4°C until they are processed for fixation in order to maintain their biological transcriptional state. However, samples should be processed at room temperature for the magnet steps detailed in the EasySep™ protocol.
Note: Please check the Parse Support Suite for the most up-to-date User Guides.
For processing Ficoll-Paque PBMCs (with trace amounts of RBCs), we recommend:
- EasySep™ RBC Depletion Reagent, from Stemcell Technologies, Catalog # 18170 EasySep™ RBC Depletion Reagent
- We use this with the magnet “The Big Easy” also from Stemcell Technologies, catalog #18001 The Big Easy" EasySep™ Magnet
The SOP that Parse Biosciences recommends:
Steps that are not explicitly in the protocol but should be performed:
1. Aliquot 1-5 mL of Ficoll-Paque PBMC media to a 15 mL tube
2. Add EDTA to a total concentration of 6 mM EDTA in media and follow the EasySep™ protocol as written. If you started with 2 mL of sample, then there should be ~ 4 mL of purified enriched cell solution.
3. Spin enriched cell solution at 200xg for 10 mins at 4°C, resuspend in 100 μL of Cell Prefixation Master Mix, and dilute 1:2 to count cells
4. Finally, move up to 1 million cells into the Cell Fixation process and top up to the correct volume of Cell Prefixation Master Mix using the current version of the Cell Fixation User Guide.
Note: We DO NOT recommend using RBC lysis for processing whole blood. RBC lysis can damage the cell membrane of PBMCs and lead to poor scRNA-seq data quality.
If you are interested in adding your protocol to our sample preparation protocols page, please reach out to us at support@parsebiosciences.com.
PDF version of protocol: