Jul 23, 2020
Version 1
PBMC- 01a - Isolation of Human PBMC from Buffy Coat V.1
- Marco Cosentino1,
- Elisa Storelli1,
- Alessandra Luini1,
- Massimiliano LM Legnaro1,
- Emanuela Rasini1,
- Marco Ferrari1,
- Franca Marino1
- 1Center for Research in Medical Pharmacology, University of Insubria (Varese, Italy)
Protocol Citation: Marco Cosentino, Elisa Storelli, Alessandra Luini, Massimiliano LM Legnaro, Emanuela Rasini, Marco Ferrari, Franca Marino 2020. PBMC- 01a - Isolation of Human PBMC from Buffy Coat. protocols.io https://dx.doi.org/10.17504/protocols.io.biw2kfge
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: July 23, 2020
Last Modified: July 23, 2020
Protocol Integer ID: 39610
Keywords: PBMC, Buffy Coat, Neuroimmune-Pharmacology, Parkinson's Disease, Cell isolation, Primary cell culture,
Abstract
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
Fetal bovine serum (FBS)BioWestCatalog #S181B-500
Ficoll Paque PLUSGe HealthcareCatalog #17144003-500 ml
RPMI 1640EuroCloneCatalog #ECM 0495L- 500 ml
Trypan Blue Solution 0.4%Thermo Fisher ScientificCatalog #15250061
Instrumentation required:
- Laminar flow hood
- Optical Microscope (manual cell count)
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 sample from buffy coat into a 50 ml conical tube.
Add an equal volume of PBS 1X and mix well.
Document
NAME
SOLUTION- 02 - Phosphate Buffered Saline (PBS)
CREATED BY
Elisa Storelli
Place 3 mL of FICOLL in a 15 mL conical tube.
CAREFULLY layer 12 mL of diluted blood on the 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 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 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 300 x g, 00:10:00 at RT.
Document
NAME
SOLUTION- 05 - Wash solution (PBS/FBS) for PBMC
CREATED BY
Elisa Storelli
Equipment
Allegra AVANTI 30
NAME
Centrifuge
TYPE
Beckman Coulter
BRAND
Beckman Italy
SKU
Remove supernatants, resuspend pellet in 1 mL of Lysis Buffer and add another 9 mL of Lysis Buffer. Immediately centrifuge the tubes at 100 x g, 00:10:00 at RT.
Document
NAME
SOLUTION- 06 - Lysis Buffer
CREATED BY
Elisa Storelli
Equipment
Allegra AVANTI 30
NAME
Centrifuge
TYPE
Beckman Coulter
BRAND
Beckman Italy
SKU
Remove supernatant and resuspend pellet in 10 mL PBS/FBS 2% and centrifuge at 300 x g, 00:10:00 at RT.
Document
NAME
SOLUTION- 05 - Wash solution (PBS/FBS) for PBMC
CREATED BY
Elisa Storelli
Equipment
Allegra AVANTI 30
NAME
Centrifuge
TYPE
Beckman Coulter
BRAND
Beckman Italy
SKU
Remove supernatant and resuspend the obtained pellet in 10 mL of RPMI/FBS 10% for cell counting.
Document
NAME
SOLUTION- 04 - Wash solution (RPMI/FBS) for PBMC
CREATED BY
Farmacologia Medica
For manual cell count use Türk solution for checking purity.
Mix 10 µl of cell suspention with an equal amount of Türk solution (dilution factor = 2), allow mixture 3 min at room temperature.
Take 10 µl of the mixture and place it inside a Bürker chamber and view under an optical microscope using 40X magnification.
Count the cells in each square found in the four corners and in the central square (see figure 1 below), including those
that lie on the bottom and left-hand perimeters, but not those that lie on the top and right hand perimeters (see figure
2 below).
Total number of cells per ml = mean number of cells x dilution factor x 104 (hemacytometer volume).
Figure 1
The gridded area of the chamber consists of nine 1 mmq squares. These squares are subdivided in three directions; 0.0625 mmq, 0.05 mmq and 0.04 mmq. The central square here in Figure 1 is further subdivided into 0.0025 mmq = 1/25 mmq squares. Count cells in 5 squares as shown.
Figure 2
Concerning those cells that lay on the perimeter of the square, count following this scheme.
Document
NAME
SOLUTION- 08 - Türk solution
CREATED BY
Farmacologia Medica
OPTIONAL STEP
For automatic cell count with Cellometer machine use Trypan Blue.
The machine will calculate the n°of cells/ml and the % of viability.
Take 10 µL of cell suspention and add an equal amount of Trypan Blue. Use all the volume to place it in a counting chamber. Place the chamber inside Cellometer and count.
Equipment
Cellometer Auto T4
NAME
Automated cell counter
TYPE
Nexcelom Bioscience
BRAND
EuroClone
SKU
Document
NAME
SOLUTION- 09 - Trypan Blue solution
CREATED BY
Farmacologia Medica
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 ± 100x106 starting from 25 ml of buffy coat.