Department of Pathology, University of California, Irvine (UCI)
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Marchenko, S., Flanagan, L. Counting Human Neural Stem Cells. J. Vis. Exp. (7), e262, doi:10.3791/262 (2007).
Knowledge of the exact number of viable cells in a given volume of a cell suspension is required for many routine tissue culture manipulations, such as plating cells for immunocytochemistry or for cell transfections. This protocol describes a straightforward and fast method for differentiating between live and dead cells and quantifying the cell concentration and total cell number using a hemacytometer. This procedure first requires detaching cells from a growth surface and resuspending them in media. Next, the cells are diluted in a solution of Trypan blue (ideally to a concentration that will give 20-50 cells per quadrant) and placed in the hemacytometer. Finally, averaging the counts of viable cells in several randomly selected quadrants, dividing the average by the volume of one 1 mm2 quadrant (0.1 μl) and multiplying by the dilution factor gives the number of cells per l. Multiplying this cell concentration by the total volume in μl gives the total cell number. This protocol describes counting human neural stem/precursor cells (hNSPCs), but can also be used for many other cell types.
Passaging Human Neural Stem Cells
Note: Please refer to the Passaging Human Neural Stem Cells article on how to detach and resuspend the cells. http://www.jove.com/index/Details.stp?ID=263
Preparing the Cell Suspension for Counting
Counting Cells in the Hemacytometer
This protocol describes a straightforward and rapid way to determine the cell concentration for a variety of experiments involving cells. Using the shortcut described in 3d, one can quickly and accurately determine the cell concentration and total cell number in a given volume.
The authors would like to acknowledge Dr. Philip H. Schwartz of the National Human Neural Stem Cell Resource at the Children s Hospital, Orange County Research Institute for providing hNSPC cultures.
|Phase Contrast Hemacytometer||Tool||Hausser Scientific||02-671-54||Distributed by Fisher under the indicated catalog #|
|Trypan Blue Stain 0.4%||Reagent||GIBCO, by Life Technologies||15250-061||Distributed by Invitrogen under the indicated catalog #|
1. Marchenko, S., Flanagan, L.A. Passaging Human Neural Stem Cells. Journal of Visualized Experiments, (7). http://www.jove.com/index/Details.stp?ID=263 (22 August 2007).
2. David R. Caprette. "Using a Hemacytometer." BioEd Online. No Date. Accessed 08 Jan. 2008. http://www.bioedonline.org/slides/slide01.cfm?q=%22hemacytometer%22&dpg=3.
3. David R. Caprette. "Counting Chamber (Hemacytometer)." BioEd Online. No Date. Accessed 08 Jan. 2008, http://www.bioedonline.org/slides/slide01.cfm?q=%22hemacytometer%22&dpg=2.