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This protocol presents an accessible guide for collecting, storing, and thawing peripheral blood mononuclear cells suitable for downstream analyses and workflows like flow cytometry and RNA sequencing. Plasma and buffy coat collections are also demonstrated.
Peripheral blood mononuclear cells (PBMCs) are commonly used in biomedical research on the immune system and its response to disease and pathogens. This detailed protocol describes the equipment, supplies, and steps for isolating, cryopreserving, and thawing high-quality and highly viable PBMCs from whole blood cells suitable for downstream applications such as flow cytometry and RNA-sequencing. Protocols for processing plasma and buffy coat from the whole blood in parallel and concurrently with PBMCs are also described. This easy-to-follow step-by-step protocol, which utilizes density gradient centrifugation to isolate PBMCs, is accompanied by a checklist of supplies, equipment, and preparation steps. This protocol is suitable for individuals with any prior experience with laboratory techniques and can be implemented in clinical or research laboratories. High-quality cell viability and RNA sequencing resulted from PBMCs collected by operators with no prior laboratory experience using this protocol.
This protocol demonstrates an accessible method and workflow for the isolation of peripheral blood mononuclear cells (PBMCs) from whole blood. This protocol is especially targeted towards novice research technicians, students, and clinical lab staff with the objective of facilitating the collection and cryopreservation of PBMCs without assuming prior training in laboratory techniques.
This protocol utilizes centrifugation to separate the components of whole blood by density. Whole blood consists of four main components listed in order of decreasing density: red blood cells/erythrocytes (~45% of volume), white blood cel....
The study protocol was approved by the UCSD and KUMC Human Protections Program and conforms to the Declaration of Helsinki. All individuals provided informed consent for participation and blood collection.
1. Processing and cryopreservation of PBMCs
Following PBMC collection and cryopreservation, viability of thawed PBMCs, monocytes, and lymphocytes from 56 unique samples was assessed by flow cytometry using reagents listed in Table 4 following manufacturers' instructions (Figure 2A-F). A mean ± SD viability of PBMCs, monocytes, and lymphocytes of 94 ± 4.0%, 98 ± 1.1%, and 93 ± 5.6%, respectively, was achieved (Figure 2G). Viability measurement by Tr.......
This protocol for PBMC collection and cryopreservation has been successfully implemented by individuals with and without prior research laboratory training. In our application, FACS and RNA-sequencing of highly viable monocytes purified from stored PBMCs resulted in high-quality sequences.
A major strength of this protocol is its accessibility. The technique presented in the protocol utilizes tubes pre-packed with a solid-density gradient medium. As a result, whole blood can be collected direc.......
We'd like to thank the patients who volunteered their consent, time, and donation of blood samples. We also acknowledge Dr. Patrick Moriarty, Julie-Ann Dutton, and Mark McClellen at the Kansas University Medical Center for their collaboration and for implementing this protocol at a remote site. CY has received research support from NIH grant 1K08HL150271.
....Name | Company | Catalog Number | Comments |
1000 µL Tips | Gilson | F174501 | Approximately 3 tips needed per patient/treatment condition for PBMC/Plasma/Buffy Coat Collection |
15 mL tube | Biopioneeer | CNT-15 | To hold Solutions 2/3 2 needed per patient/treatment condition for PBMC/Plasma/Buffy Coat Collection |
2 mL Cryovials | Globe Scientific | 3012 | Approximately 40 cryovials needed per patient/treatment condition for PBMC/Plasma/Buffy Coat Collection |
20 µL Tips | Gilson | F174201 | For use in cell counting; volume needed is dependent on method of cell counting used. 1 tip is needed per 1 mL of unpooled frozen PBMCs to be defrosted |
50 mL Conical Tube | CEM Corporation | 50-187-7683 | 2 needed per patient/treatment condition for PBMC/Plasma/Buffy Coat Collection 3 needed per 1 mL of unpooled frozen PBMCs to be defrosted |
CD14 Antibody | Biolegend | 325621 | |
CD16 Antibody | Biolegend | 360723 | |
CD19 Antibody | Biolegend | 363007 | |
CD3 Antibody | Biolegend | 300405 | |
CD56 Antibody | Biolegend | 318303 | |
CD66b Antibody | Biolegend | 305103 | |
Cell Strainer | Biopioneeer | DGN258367 | 1 needed per 1 mL of unpooled frozen PBMCs to be defrosted |
CPT Mononuclear Cell Preparation Tube | BD Biosciences | 362753 | 6 tubes needed per patient/treatment condition for PBMC/Plasma/Buffy Coat Collection |
Cryo Freezer Box | Southern Labware | SB2CC-81 | Holds 81 tubes 1 needed per patient/treatment condition for PBMC/Plasma/Buffy Coat Collection |
Cryotube Rack | Fisherbrand | 05-669-45 | Holds up to 50 cryovials |
DMSO | Invitrogen | 15575020 | Approximately 2.5 mL needed per patient/treatment condition for PBMC/Plasma/Buffy Coat Collection |
DNase/RNase-Free Distilled Water | Invitrogen | 10977015 | Approximately 2 mL needed per patient/treatment condition for PBMC/Plasma/Buffy Coat Collection Approximately 9 mL needed per 1 mL of frozen PBMCs to be defrosted |
EDTA Tubes | BD Biosciences | 366643 | 1 tube needed per patient/treatment condition for PBMC/Plasma/Buffy Coat Collection |
Flavopiridol | Sigma-Aldrich | F3055-5MG | |
HLA-DR Antibody | Biolegend | 307609 | |
Human Serum Albumin | GeminiBio | 800-120 | Approximately 25 mL needed per patient or treatment condition for PBMC/Plasma/Buffy Coat Collection |
Human Trustain FcX | Biolegend | 422301 | |
Isopropanol | Sigma-Aldrich | W292912-1KG-K | For use in Mr. Frosty |
Label Printer | Phomemo | M110-WH | |
Live-Dead Stain | Biolegend | 423105 | |
Mr. Frosty | Thermo Scientific | 5100-0001 | Holds up to 18 tubes. 2 Mr. Frostys needed per patient/treatment condition for PBMC/Plasma/Buffy Coat Collection |
Multidispense Pipette | Brandtech | 705110 | |
Multidispense Pipette Tips | Brandtech | 705744 | Approximately 3 tips needed per patient/treatment condition for PBMC/Plasma/Buffy Coat Collection |
P1000 Pipette | Gilson | F144059M | |
P20 Pipette | Gilson | F144056M | For use in cell counting; volume needed is dependent on method of cell counting used. |
Printer Labels | Phomemo | PM-M110-3020 | Approximately 45 labels needed per patient/treatment condition for PBMC/Plasma/Buffy Coat Collection Approximately 5 labels needed per 1 mL of unpooled frozen PBMCs to be defrosted |
RNase-Free EDTA (0.5 M) | Invitrogen | AM9260G | Approximately 40 µL needed per 1 mL of frozen PBMCs to be defrosted |
RNase-Free PBS (10X) | Invitrogen | AM9625 | Approximately 1 mL needed per 1 mL of frozen PBMCs to be defrosted |
RNasin | Promega | N2111 | |
RPMI | Corning | 10-040-CV | Approximately 80 mL needed per patient or treatment condition for PBMC/Plasma/Buffy Coat Collection |
Transfer Pipettes | Fisherbrand | 13-711-7M | Approximately 5 needed per patient/treatment condition for PBMC/Plasma/Buffy Coat Collection |
Tube Holder | Endicott-Seymour | 14-781-15 | Holds up to 80 CPT/EDTA Tubes |
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