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

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Separation and purification of PBMC from BUFFY COAT: list of published work using this protocol</span></div><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;"><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></li><li style = "counter-reset:ol0;list-style-type:disc;"></li><li style = "counter-reset:ol0;list-style-type:disc;"><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></li><li style = "counter-reset:ol0;list-style-type:disc;"></li><li style = "counter-reset:ol0;list-style-type:disc;"><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></li></ul></div><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;"><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></li><li style = "counter-reset:ol0;list-style-type:disc;"></li><li style = "counter-reset:ol0;list-style-type:disc;"><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></li></ul></div><div class = "text-block"><ul style = "list-style-type:disc;"></ul></div></div>

[STEPS]
?. 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.
{"blocks":[{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"7sj4k","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"2mf15","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"323f7","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"861u4","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"6lket","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"8nmm6","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"6imgb","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"1odal","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"7412g","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"7322o","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"40qj6","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"93nhp","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"dh88e","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"25hl2","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"fn89i","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"90dau","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"4etvc","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"276vd","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"5c9js","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"67foe","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"41hs4","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"b2imh","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"e6rki","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"e0orh","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"c5t09","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"96bhv","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"4eehe","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"4j54d","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"bulv7","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"72igt","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"fl6ge","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"a0st4","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"8toa","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"c8j2o","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"bmve4","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"ba0p9","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"5e8jd","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"apavl","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"b987t","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"dbhvn","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"drtn1","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"cl0ml","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"6jst4","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"b32nu","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"eajou","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"14nuu","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"crtl2","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"5alud","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"efrgk","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"9jcq8","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"a0mut","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"d4lg9","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"1ubu4","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"2s48n","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"7sb2u","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"9v5gl","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"dp6d6","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"14b2q","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"d8hv4","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"e7fj","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"4n7un","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"c03m7","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"7p76l","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"72ktr","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"bs4ub","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"dvvd6","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"5u1gq","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"bkp04","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"dr8rm","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"b9rrp","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"8n9j1","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"1nd2g","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"b04cu","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"6alh5","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"4sl","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"9fshf","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"24b3r","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"4n2i1","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"a57l0","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"915pq","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"fs7ch","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"erhan","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"pj5e","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"a9qp","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"cpo39","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"3pt9n","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"fvmp0","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"cn5j0","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"7f760","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"2or5s","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"aec3q","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"3on6v","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"fgc1s","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"6h1g9","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"7g56n","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"ea82p","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"f7ki8","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"717c8","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"fjr80","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"di7og","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"doipp","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"17cao","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"f843c","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"dbjpu","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"5pna4","text":"","type":"unstyled"},{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[],"key":"2h0h9","text":"","type":"unstyled"}],"entityMap":[]}
?. Place  of FICOLL in a  conical tube.
3 ml
15 ml
?. CAREFULLY  layer  of diluted blood on the FICOLL with a glass Pasteur Pipette to a final volume of 15 ml as shown in the figure below.
12 ml
?. Centrifuge samples  (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 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  at RT.
Centrifuge: 300 34, 10 min
?. Remove supernatants, resuspend pellet in  of Lysis Buffer and add another  of Lysis Buffer. Immediately centrifuge the tubes at at RT.
1 ml
9 ml
Centrifuge: 100 34, 10 min
?. Remove supernatant and resuspend pellet in PBS/FBS 2% and centrifuge at   at RT.
10 ml
Centrifuge: 300 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.
10 µl
10 µl
?. 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.
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.
?. 
?. 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.