TITLE: CELL COUNT- 01 - Manual cell count with Trypan Blue
AUTHORS: Marco Cosentino, Elisa Storelli, Alessandra Luini, Massimiliano Legnaro, Emanuela Rasini, Marco Ferrari, Franca Marino

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">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., Struchio A., Mauri M., Bono G., Marino F., Cosentino M. 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 (2018). 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></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 class = "text-block">- Boydum A.Isolation of mononuclear cells and granulocytes from human blood. Scand.J.Clin.Lab. Invest. 21 (Suppl.97): 77-89, 1968</div><div class = "text-block">- Alex Mabou Tagne, Franca Marino, Massimiliano Legnaro, Alessandra Luini, Barbara Pacchetti and Marco Cosentino. A Novel Standardized Cannabis sativa L. Extract and Its Constituent Cannabidiol Inhibit Human Polymorphonuclear Leukocyte Functions. Int J Mol Sci2019 Apr; 20(8): 1833. Published online 2019 Apr 13. doi: 10.3390/ijms20081833.</div><div class = "text-block">- Angela Scanzano, Laura Schembri, Emanuela Rasini, Alessandra Luini, Jessica Dallatorre, Massimiliano Legnaro, Raffaella Bombelli, Terenzio Congiu, Marco Cosentino, Franca Marino. Adrenergic Modulation of Migration, CD11b and CD18 Expression, ROS and interleukin-8 Production by Human Polymorphonuclear Leukocytes. Inflamm Res. 2015 Feb;64(2):127-35. doi: 10.1007/s00011-014-0791-8. Epub 2015 Jan 6.</div></div>

[STEPS]
?. Use Trypan Blue solution for checking cell viability.Mix   of cell suspention with an equal amount of Trypan Blue solution (dilution factor = 2).
10 µl
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?. Take of the mixture and place it inside a Bürker chamber and view under an optical microscope using 40X magnification.
10 µl
?. 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).Figure 1The 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 2Concerning those cells that lay on the perimeter of the square, count following this scheme.