Aug 31, 2022
Isolation of PBMCs From Whole Blood
- 1IRCCS Mondino Foundation, Pavia
Protocol Citation: Gerardo Ongari 2022. Isolation of PBMCs From Whole Blood. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlkwbm6l5r/v1
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: August 31, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 69397
Keywords: ASAPCRN
Abstract
This protocol details methods for the isolation of peripheral blood mononuclear cells (PBMCs) from Whole Blood
Materials
Materials
• 4 EDTA Lavender top tubes (9mL/each)
• Density gradient medium (Histopaque-1077 Sigma-Aldrich)
• Phosphate Buffered Saline (DPBS without calcium without magnesium)
• Tubes for centrifugations
• 1.8 ml Cryotube vials (Nunc 055004)
Safety warnings
Please refer to the Safety Data Sheets (SDS) for health and environmental hazards.
Before start
Pre-procedure: Ensure all reagents are at room temperature (15 - 25°C). The procedure must be carried out under a cell culture hood.
Step 1: Plasma Isolation
Step 1: Plasma Isolation
Collect 36 mL whole blood into EDTA tubes
Centrifuge the blood samples at 1000 x g, Room temperature, 00:20:00
20m
Carefully remove collection tubes from centrifuge; plasma is the top layer above the rest of the whole blood layer
Using caution not bring up the remaining whole blood layer, remove plasma and aliquot it into 1.5 mL microcentrifuge tubes
Centrifuge the isolated plasma at 1600 x g, Room temperature, 00:20:00 , to remove residual cells and debris
20m
After centrifugation, aliquot the plasma supernatant into 1mL aliquots by using microcentrifuge tubes labeled with patient ID
Freeze at -80 °C .
Step 2: PBMC isolation
Step 2: PBMC isolation
1h
1h
Dilute the rest of the whole blood sample to a 1:1 volume ratio with the PBS
Add 15 mL density gradient medium to a fresh 50mL conical tube and gently layer the diluted blood on top of the density gradient medium. Take care not to mix the two layers.
Centrifuge at 800 x g, Room temperature, 00:20:00 , with brake OFF
20m
Carefully harvest the cells by inserting the pipette directly through the upper plasma layer to the mononuclear cells at the interface. Alternatively, you can first remove the upper layer and then collect the cells
Wash the harvested PBMC in 12 mL of PBS
Centrifuge at 300 x g, Room temperature, 00:15:00
15m
Discard the surnatant and wash the PBMC pellet in 5mL of PBS
Centrifuge at 300 x g, Room temperature, 00:15:00
15m
Discard the surnatant and resuspend the cell pellet in 5 mL of PBS
Count the cells using Trypan blue staining.
Resuspend PBMCs to 5x106 cells/mL in PBS and aliquot them in cryotube vials (1mL/vial) labeled with patient ID
Centrifuge at 800 x g, Room temperature, 00:10:00
10m
Discard the surnatant and store the cell pellets at -80 °C