Oct 30, 2020
Purification of PBMCs
- Roser Vento-Tormo1
- 1Wellcome Sanger Institute, Cambridge, UK
- Human Cell Atlas Method Development Community
- Coronavirus Method Development Community
Protocol Citation: Roser Vento-Tormo 2020. Purification of PBMCs. protocols.io https://dx.doi.org/10.17504/protocols.io.bjinkkde
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 10, 2020
Last Modified: February 03, 2021
Protocol Integer ID: 40238
Keywords: PBMC, COVID-19, purification,
Abstract
This protocol details the steps and methods of PBMC purification
Attachments
Materials
- Ficoll
- Blood tubes
- 70% ethanol
- 50 mL conical tube(s)
- PBS
- serological pipette
- centrifuge
- pipet tips
- FBS + 10% DMSO
Safety warnings
For hazard and safety information, please see the Safety Data Sheet (SDS).
Ficoll Density Gradient Centrifugation
Ficoll Density Gradient Centrifugation
Bring Ficoll to Room temperature .
Clean the blood tubes with 70% ethanol and transfer the blood to a 50mL tube in a BSL2 hood.
Dilute blood 1:1 in PBS and mix carefully.
Place 15 mL Ficoll in each of the 50mL conical tubes.
Cover the Ficoll with the blood/PBS mixture using a serological pippette at the lowest speed.
Note
To avoid compromising the purity of the PBMC, Ficoll and blood should not be mixed in any way. It is therefore best to hold the tube at 45º angle and the blood mixture should exit the pipette and run along the wall of the tube very slowly.
Centrifuge the samples at 800 x g, 20°C, 00:30:00 , selecting acceleration/deceleration rates of 9/1.
Note
Normally the rotor brakes have to be fully deactivated to effectively prevent phase mixing. It is also very important to strictly adhere to the specified temperatures because temperature differences will change the density rates of the fluids and may have a negative impact on the separation results.
After completion of the centrifugation, remove the samples from the centrifuge to avoid phase mixing.
Lymphocyte Purification
Lymphocyte Purification
Carefully aspirate 2/3 of the top layer (containing the plasma and platelets) using a sterile serological pipette until the interface (containing the mononuclear cells) is within reach.
Using a pipette, aspirate the entire lymphocyte layer while keeping the volume minimal and transfer to a clean tube.
Note
Try to transfer as little Ficoll and supernatant as possible during this step.
Add at least 3 volumes of PBS to the lymphocyte layer and mix very carefully by pipetting up and down.
Centrifuge at 100 x g, 20°C, 00:10:00 and remove the supernatant.
Add at least 3 volumes of PBS to the lymphocyte layer and mix very carefully by pipetting up and down.
Centrifuge at 100 x g, 20°C, 00:10:00 and remove the supernatant.
Resuspend the cells in FBS + 10% DMSO and cryopreserve in liquid nitrogen.