Name: Counting Human Neural Stem Cells (Video) | JoVE Journal
Uploaded: 2007-08-22T00:00:00.000+00:00
Duration: 6 min 37 s
Description: Learn to count human neural stem cells using a hemacytometer and trypan blue dye for accurate viability assessment. This protocol enables precise quantification for tissue culture experiments.

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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.

Passaging Human Neural Stem Cells
Note: Please refer to the <a href="https://www.jove.com/index/details.stp?ID=263">Passaging Human Neural Stem Cells</a> article on how to detach and resuspend the cells. https://www.jove.com/index/Details.stp?ID=263
Preparing the Cell Suspension for Counting
<ol>
<li>Place 25 µl of 0.4% Trypan Blue solution and 20 µl of cell media into an eppendorf tube.</li>
<li>Resuspend the cells to be counted by gently tapping the tube containing the cells in a known volume of media to create a homogenous suspension. Place 5 µl of cells into the eppendorf tube from the previous step. This step dilutes the cell concentration by a factor of 10.</li>
<li>Place the cover glass on the hemacytometer, and load about 11-12 µl of the cell suspension. <img src="/files/ftp_upload/old/262_2008_7_18_4/262_Figure-01.png" alt="Hemocytometer diagram; top and side views for cell counting with grid areas and sample depth 0.1 mm." width="511" height="139" data-altupdatedat="1761735611000" data-alt="262_Figure-01"> </li>
</ol>
Counting Cells in the Hemacytometer
<ol>
<li>Observe the entire grid of the hemacytometer in a phase contrast microscope. Focus on one quadrant of the grid (such as the one in red). <img src="/files/ftp_upload/old/262_2008_7_18_5/262_Figure-02.png" alt="Hemacytometer grid diagram with magnified views for cell counting, square size detail shown." width="492" height="278" data-altupdatedat="1761735611000" data-alt="262_Figure-02"> </li>
<li>Dead or dying cells will appear blue because their membrane has been damaged and are not able to exclude the Trypan blue dye. Viable cells will not appear blue and will be surrounded by a “halo” of light in phase contrast (will be “phase-bright”). </li>
<li>Count the number of viable cells in one quadrant (area 1 mm2). You can also determine cell viability by dividing the number of viable cells by the total number of cells. Count cells in 3-4 random quadrants and determine the average number of cells per quadrant. Since the volume of a quadrant is known to be 10-4 ml or 0.1 µl, the concentration of cells can be determined <img src="/files/ftp_upload/old/262_2008_7_18_6/262_Figure-03.png" alt="Cell counting setup; formula: quadrant volume, dilution factor; hemocytometer diagram." width="741" height="165" data-altupdatedat="1761735611000" data-alt="262_Figure-03"> </li>
<li>You can use the following shortcut to determine the #cells/µl: <img src="/files/ftp_upload/old/262_2008_7_18_7/262_Figure-04.png" alt="Cell concentration calculation formula diagram, #cells/quadrant x10 x10=#cells/µl" width="368" height="69" data-altupdatedat="1761735611000" data-alt="262_Figure-04"> Total # of cells in tube =# cells/µl X µl in tube</li>
</ol>

<ol><li>Marchenko, S., Flanagan, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Passaging+Human+Neural+Stem+Cells%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Passaging Human Neural Stem Cells.</a> Journal of Visualized Experiments. 7, 
 
 <a target="_blank" href="http://www.jove.com/index/Details.stp?ID=263">http://www.jove.com/index/Details.stp?ID=263</a> (2007).</li>
<li>Caprette, D. avidR. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Using+a+Hemacytometer%5BTitle%5D)+AND+%22BioEd+Online%22%5BJournal%5D)">Using a Hemacytometer.</a> BioEd Online. , 
 
 <a target="_blank" href="http://www.bioedonline.org/slides/slide01.cfm?q=%22hemacytometer&dpg=3">http://www.bioedonline.org/slides/slide01.cfm?q=%22hemacytometer&dpg=3</a> (2008).</li>
<li>Caprette, D. avidR. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Counting+Chamber+%28Hemacytometer%29%5BTitle%5D)+AND+%22BioEd+Online%22%5BJournal%5D)">Counting Chamber (Hemacytometer).</a> BioEd Online. , 
 
 <a target="_blank" href="http://www.bioedonline.org/slides/slide01.cfm?q=%22hemacytometer%22&dpg=2
">http://www.bioedonline.org/slides/slide01.cfm?q=%22hemacytometer%22&dpg=2
</a> (2008).</li>
</ol>

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.

<table><tbody><tr><td>Phase Contrast Hemacytometer</td><td>Tool</td><td>Hausser Scientific</td><td>02-671-54</td><td>Distributed by Fisher under the indicated catalog #</td></tr><tr><td>Trypan Blue Stain 0.4%</td><td>Reagent</td><td>GIBCO, by Life Technologies</td><td>15250-061</td><td>Distributed by Invitrogen under the indicated catalog #</td></tr></tbody></table>

counting human neural stem cells

Counting Human Neural Stem Cells

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 &mu;l) and multiplying by the dilution factor gives the number of cells per l. Multiplying this cell concentration by the total volume in &mu;l 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.

Knowledge of the exact number of viable cells is required for many tissue culture manipulations. This protocol describes how to differentiate between live and dead cells and quantify cells using a hemacytometer. Although it describes counting human neural stem/precursor cells (hNSPCs), it can be used for other cell types.

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 ...

Now I'm going to show you how to count human neuro precursor cells and you would count your cells when you want to put them for immuno chemistry or when you want to do a transfection with nuclear factor machine. I use a phase contrast hemo cytometer that has a, an edge grid and also we use a dye called trip and blue. And the dye is useful in that it can differentiate between live and dead cells.Dead or dying cells take up this dye and they appear blue. When you look at the cells in the hemo, cytometer and live cells are able to exclude this dye and they, they, they don't appear blue. So that way you can determine the viability of your cells to prepare a solution of trip and blue and media.I'm going to add 20 microliters of trap and blue solution into this 0.5 mil epi tube. And then I'm going to add 25 microliters of media. So that way right now I have 45 microliters of, of a trap pan blue and media solution so that when I add five microliters of cell suspension, I'm going to have a one to 10 dilution.And so now I'm, I'm going to add five microliters of cell suspension. First I'm going to make sure cells are well resuspended by finger vortexing. So now I want to make sure this solut

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Learn to count human neural stem cells using a hemacytometer and trypan blue dye for accurate viability assessment. This protocol enables precise quantification for tissue culture experiments.

Counting Human Neural Stem Cells

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