Nov 21, 2020
Version 4
PBMC- 01b Isolation of human PBMC from Whole Blood V.4
- Marco Cosentino1,
- Elisa Storelli2,
- Alessandra Luini1,
- Massimiliano Legnaro3,
- Emanuela Rasini3,
- Marco Ferrari3,
- Franca Marino3
- 1Center for Research in Medical Pharmacology, University of Insubria (Varese, Italy);
- 2Center for Research in Medical Pharmacology, University of Insubria;
- 3University of Insubria
Protocol Citation: Marco Cosentino, Elisa Storelli, Alessandra Luini, Massimiliano Legnaro, Emanuela Rasini, Marco Ferrari, Franca Marino 2020. PBMC- 01b Isolation of human PBMC from Whole Blood. protocols.io https://dx.doi.org/10.17504/protocols.io.bpxmmpk6Version created by Farmacologia Medica
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: November 21, 2020
Last Modified: November 21, 2020
Protocol Integer ID: 44749
Keywords: PBMC, Fresh Blood, Neuroimmune-Pharmacology, Parkinson's Disease, Cell isolation, Primary cell culture, isolation of human pbmc, human pbmc, patients with parkinson, purification of pbmc, parkinson, pbmc, influence of dopamine receptor gene polymorphism, dopaminergic receptor, dopamine receptor gene polymorphism, dopaminergic receptors on cd4, journal of neuroinflammation, whole blood separation, treg in drug,
Abstract
Separation and purification of PBMC from FRESH BLOOD: list of published work using this protocol
Kustrimovic, N., Comi, C., Magistrelli, L., Rasini, E., Legnaro, M., Bombelli, R., Aleksic, I., Blandini, F., Minafra, B., Riboldazzi, G., Sturchio, A., Mauri, M., Bono, G., Marino, F., & Cosentino, M. (2018). Parkinson's disease patients have a complex phenotypic and functional Th1 bias: cross-sectional studies of CD4+ Th1/Th2/T17 and Treg in drug-naïve and drug-treated patients. Journal of neuroinflammation, 15(1), 205. https://doi.org/10.1186/s12974-018-1248-8
Kustrimovic, N., Rasini, E., Legnaro, M., Bombelli, R., Aleksic, I., Blandini, F., Comi, C., Mauri, M., Minafra, B., Riboldazzi, G., Sanchez-Guajardo, V., Marino, F., & Cosentino, M. (2016). Dopaminergic Receptors on CD4+ T Naive and Memory Lymphocytes Correlate with Motor Impairment in Patients with Parkinson's Disease. Scientific reports, 6, 33738. https://doi.org/10.1038/srep33738
Cosentino M., Ferrari M., Kustrimovic N., Rasini E., Marino F. (2015). Influence of dopamine receptor gene polymorphisms on circulating T lymphocytes: A pilot study in healthy subjects. Human immunology, 76, 10, 747-752. https://doi.org/10.1016/j.humimm.2015.09.032
Materials
MATERIALS
Ficoll Paque PLUSGE HealthcareCatalog #17144003-500 ml
Fetal Bovine Serum (FBS)EuroCloneCatalog #ECS0180L-500 ml
RPMI 1640EuroCloneCatalog #ECM 0495L- 500 ml
Trypan Blue solution 0.4%Merck MilliporeSigma (Sigma-Aldrich)Catalog #T8154- 100 ml
NaClMerck MilliporeSigma (Sigma-Aldrich)Catalog #S9625
Na2HPO4*7H2OMerckCatalog #1.06574.1000
NaH2PO4MerckCatalog #1.06346.0500
NH4ClMerckCatalog #1.01145.1000
KHCO3MerckCatalog #1.04854.500
EDTAMerck MilliporeSigma (Sigma-Aldrich)Catalog #ED2SS
Acetic Acid 100%Merck MilliporeSigma (Sigma-Aldrich)Catalog #A6283
Gentian violet 1%Marco VitiCatalog #not available
Instrumentation required:
- Laminar flow hood
- Autoclave
Troubleshooting
Before start
If you need to obtain PBMC for cell culture, make sure you are using sterile PBS, culture medium, filtered Lysis Buffer and sterile plastic disposables as well. Moreover, work under laminar flow hood when you are processing samples. Otherwise, use non-sterile solutions and plastic disposables, and process samples in cell isolation laboratory.
ALL REAGENTS USED IN THIS PROTOCOL MUST BE AT ROOM TEMPERATURE!
Put the needed amount of blood sampl into a 50 mL conical tube.
Add an equal volume of PBS 1X and mix well.
Place 3 mL of FICOLL in a 15 mL conical tube.
Carefully layer 12 mL of diluted blood on FICOLL with a glass Pasteur Pipette to a final volume of 15 ml as shown in the figure below.
Centrifuge samples 400 x g, 00:40:00 at room temperature (RT) without break.
Equipment
Allegra AVANTI 30
NAME
Centrifuge
TYPE
Beckman Coulter
BRAND
Beckman Italy
SKU
After centrifugation, take out the tubes carefully to not disturb the mononuclear cell layer that appears as a white, cloudy band between the plasma and FICOLL as shown in the figure below.
Carefully with a glass Pasteur pipette transfer the mononuclear lymphocyte cell layer to another 15 ml conical tube.
Wash the isolated PBMC with PBS/FBS 2% to a final volume of 10 mL and centrifuge at
600 x g, 00:10:00 at RT.
Remove supernatants, resuspend pellet in 1 mL of Lysis Buffer and add another 9 mL of Lysis Buffer. Immediately centrifuge tubes at 300 x g, 00:10:00 at RT.
Remove supernatant and resuspend pellet in 10 mL of PBS/FBS 2% and centrifuge at 600 x g, 00:10:00 at RT.
Remove supernatant and resuspend the obtained pellet in 10 mL of RPMI/FBS 10% for cell counting.
For manual cell count use Türk solution for checking purity.
Follow protocol CELL COUNT- 02.
OPTIONAL STEP
For automatic cell count with Cellometer machine use Trypan Blue.
Follow protocol CELL COUNT- 03.
If needed, check the purity of PBMC suspension by using morphological parameter of the flow cytometer.
For this test 0.5x106 PBMC in 500 µl of PBS are enough.
Equipment
BD FACS Celesta
NAME
Flow Cytometer
TYPE
Becton Dickinson
BRAND
Milan Italy BD
SKU
Expected results
Expected result
VIABILITY - The expected viability by Trypan Blue should be ≥90 %.
PURITY - The PBMC suspension obtained should contain at least 80% of lymphocytes, 10-15% of monocytes and few contaminant PMN cells (≤ 5%) as confirmed by flow cytometry.
YIELD - The expected amount of PBMCs should be ± 28,5x106 starting from 25 ml of fresh blood.