Apr 28, 2020

Public workspaceSeparation and purification of human PBMC from BUFFY COAT V.1

DOI
dx.doi.org/10.17504/protocols.io.bc5kiy4w
  • 1Center for Research in Medical Pharmacology, University of Insubria (Varese, Italy);
  • 2University of Insubria
Elisa Storelli
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DOI: dx.doi.org/10.17504/protocols.io.bc5kiy4w
Protocol Citation: Marco Cosentino, Elisa Storelli, Alessandra Luini, Massimiliano LM Legnaro, Emanuela Rasini, Marco Ferrari, Franca Marino 2020. Separation and purification of human PBMC from BUFFY COAT. protocols.io https://dx.doi.org/10.17504/protocols.io.bc5kiy4w
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: March 02, 2020
Last Modified: April 28, 2020
Protocol Integer ID: 33676
Keywords: PBMC, Buffy Coat, Neuroimmune-Pharmacology, Parkinson's Disease, Cell isolation, Primary cell culture,
Abstract
Separation and purification of PBMC from BUFFY COAT: 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
ReagentFicoll Paque PLUSGe HealthcareCatalog #17144003-500 ml
ReagentFetal Bovine Serum (FBS)EuroCloneCatalog #ECS0180L-500 ml
ReagentRPMI 1640EuroCloneCatalog #ECM 0495L- 500 ml
ReagentTrypan Blue solution 0.4%Sigma AldrichCatalog #T8154- 100 ml
Instrumentation required:

  • Laminar flow hood
  • Centrifuge
  • Cellometer (automated cell counter) or Optical Microscope (manual cell count)
  • Flow Cytometer
  • Autoclave
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
PBS 1X
NAME

PBS 1X

CREATED BY
Marco Ferrari



Place Amount3 mL of FICOLL in a Amount15 mL conical tube.


CAREFULLY layer Amount12 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.




Critical
Centrifuge samples Centrifigation400 x g, 00:40:00 (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 mononuclear lymphocyte cell layer to another 15 ml conical tube.
Critical
Wash the isolated PBMC with PBS/FBS 2% to a final volume of 10 ml and centrifuge at Centrifigation300 x g, 00:10:00 at RT.

Document
RPMI - FBS and PBS - FBS
NAME

RPMI - FBS and PBS - FBS

CREATED BY
Elisa Storelli

Remove supernatants, resuspend pellet in Amount1 mL of Lysis Buffer and add another Amount9 mL of Lysis Buffer. Immediately centrifuge the tubes atCentrifigation100 x g, 00:10:00 at RT.

Document
Lysis Buffer
NAME

Lysis Buffer

CREATED BY
Elisa Storelli

Remove supernatant and resuspend pellet in Amount10 mL PBS/FBS 2% and centrifuge at Centrifigation300 x g, 00:10:00 at RT.


Document
RPMI - FBS and PBS - FBS
NAME

RPMI - FBS and PBS - FBS

CREATED BY
Elisa Storelli


Remove supernatant and resuspend the obtained pellet in Amount10 mL of RPMI/FBS 10% for cell counting.

Document
RPMI - FBS and PBS - FBS
NAME

RPMI - FBS and PBS - FBS

CREATED BY
Elisa Storelli

For manual cell count use Türk solution for checking purity.

Mix Amount10 µL of cell suspention with an equal amount of Türk solution (dilution factor = 2), allow mixture 3 min at room temperature.
Take Amount10 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

Figure 2


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 Amount10 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



Optional
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 FACSCelesta
NAME
Flow Cytometer
TYPE
Becton Dickinson, Milan Italy
BRAND
BD
SKU

Optional

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.