TITLE: Dilution of the Cell Suspension
AUTHORS: Nicolas Giguère

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol outlines the requirements for successful dilution of cell suspension.</div></div>

[STEPS]
?. [Dilution of the Cell Suspension]
Re-suspend the cell pellet in .
[trituration solution]
?. [Dilution of the Cell Suspension]
Take  of the suspension and dilute it in .
10 µl
[Trypan Blue Solution (Gibco 15250061)]
?. [Dilution of the Cell Suspension]
Take  of this mix to count on the hematocytometer.
10 µl
?. [Dilution of the Cell Suspension]
Count the living cells and also the number of dead cells (colored in blue-black, the number should be less than 20% of the total number of cells).
?. [Dilution of the Cell Suspension]
V \text { to add }=\left[\left(\frac{n b \text { of cells counted } \times \text { dilution factor}}{\text {dial volume } \times \text { seeding concentration}}\right) \times \text { cell suspension volume }\right]-\text { cell suspension volume }The volume of solution covering 1 millimeter squared of the hematocytometer (see the "How to Count the Cells" section) is 0.1 µl and the dilution factor is 2. If the concentration required for seeding is 240,000 cells/ml, the volume of trituration solution to be added to the 490 µl of cell suspension is:V \text { to add }=\left[\left(\frac{n b \text { of cells counted } \times 2}{0.0001 \mathrm{ml} \times 240 \text { 000 cells } / \mathrm{ml}}\right) \times 0.490 \mathrm{ml}\right]-0.490 \mathrm{ml}
?. [How to Count the Cells]
To count your cell sample, dilute  in  and then load the hematocytometer.
[of your sample]
[Trypan Blue Solution (Gibco 15250061)]
?. [How to Count the Cells]
The counting chamber is covered with a laser engraved grid. Quadrant lines make counting easier. Choose the appropriate quadrant size based on the density of the cells in your sample. For example, for a small number of cells (less than 20 in a quadrant of 16), count all the cells in the 4 quadrants of 16, and divide this number by 4. For samples with a quantity of more than 20 cells, choose the diagonal in the 4 quadrants of 16.
?. [How to Count the Cells]
Regardless of the area you choose or the density of the sample, count at least 20 to 50 cells per quadrant.
?. [How to Count the Cells]
Not all cells will fall perfectly into the quadrants. For example, you can count the cells that touch the top and left lines but ignore the cells that touch the bottom and right lines. Use a cell counter to keep track of the number of cells counted.
?. [How to Count the Cells]
The volume of solution covering 1 millimeter squared of the hematocytometer is 0.1 µl and the dilution factor is 2. If the concentration required for seeding is 240,000 cells/ml, the volume of trituration solution to be added to the 490 µl of cell suspension is:V \text { to add }=\left[\left(\frac{n b \text { of cells counted } \times \text { dilution factor}}{\text {dial volume } \times \text { seeding concentration}}\right) \times \text { cell suspension volume }\right]-\text { cell suspension volume }
?. [Determine Cell Viability]
While counting cells under the microscope, assess the viability of the cells in your sample.
Trypan blue is a dye used to determine viability. The living cell excludes this dye, but the dead cell has no intact membrane allowing the trypan blue to pass through and mark the cytoplasm. In phase contrast, living cells appear bright and golden and dead cells appear dull and blue. The number of dead cells should be less than 20% of the total number of cells.
?. [Determine Cell Viability]
When you have finished counting, clean the counting chamber.