TITLE: Separation and purification of human PBMC from FRESH BLOOD
AUTHORS: Marco Cosentino, Elisa Storelli, Alessandra Luini, Massimiliano Legnaro, Emanuela Rasini, Marco Ferrari, Franca Marino

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Separation and purification of PBMC from FRESH BLOOD: list of published work using this protocol</div><div class = "text-block"><span>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. </span><a href="https://doi.org/10.1186/s12974-018-1248-8" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;:UNDERLINE;">https://doi.org/10.1186/s12974-018-1248-8</span></a><span style = ":;:UNDERLINE;"></span></div><div class = "text-block"><span>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. </span><a href="https://doi.org/10.1038/srep33738" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://doi.org/10.1038/srep33738</span></a></div><div class = "text-block"><span>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. </span><a href="https://doi.org/10.1016/j.humimm.2015.09.032" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://doi.org/10.1016/j.humimm.2015.09.032</span></a></div></div>

[STEPS]
?. Put the needed amount of blood sampl into a   conical tube.
50 ml
?. Add an equal volume of PBS 1X and mix well.
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?. Place  of FICOLL in a   conical tube.
3 ml
15 ml
?. Carefully layer   of diluted blood on FICOLL with a glass Pasteur Pipette to a final volume of 15 ml as shown in the figure below.
12 ml
?. Centrifuge samples  at room temperature (RT) without break.
Centrifuge: 400 34, 40 min
?. 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   and centrifuge at at RT.
10 ml
Centrifuge: 600 34, 10 min
?. Remove supernatants, resuspend pellet in  of Lysis Buffer and add another  of Lysis Buffer. Immediately centrifuge tubes at  at RT.
1 ml
9 ml
Centrifuge: 300 34, 10 min
?. Remove supernatant and resuspend pellet in  of PBS/FBS 2% and centrifuge at   at RT.
10 ml
Centrifuge: 600 34, 10 min
?. Remove supernatant and resuspend the obtained pellet in  of RPMI/FBS 10% for cell counting.
10 ml
?. For manual cell count use Türk solution for checking purity.Mix   of cell suspention with an equal amount of Türk solution (dilution factor = 2), allow mixture 3 min at room temperature.Take 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 thosethat 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).
10 µl
10 µl
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?. OPTIONAL STEPFor automatic cell count with Cellometer machine use Trypan Blue. The machine will calculate the n° of cells/ml and the % of viability.Take  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.
10 µl
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?. 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.
?. Expected results
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 ± 28,5x10*6 starting from 25 ml of fresh blood.