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The QIAGEN Guide to Animal Cell Culture

by centrifugation at 200 x g for 5 min (suspension cell cultures). Resuspend the cells in an appropriate volume of prewarmed growth medium. At least 106 cells/ml are required for accurate counting.

Tip - It may be necessary to centrifuge cells and resuspend in a smaller volume to obtain the desired cell concentration for counting. For adherent cells, it is important to produce a single-cell suspension after trypsinizing. Cell clumping will make counting difficult and inaccurate.

4. Mix the cell suspension sample thoroughly. Using a pipet, immediately transfer 20 l to the edge of one side of the coverslip to fill one chamber of the hemocytometer. Repeat for the second chamber.

Tip- The cell distribution should be homogeneous in both chambers.

Tip - The cell suspension is drawn under the coverslip and into the chamber by capillary action. The cell suspension should just fill the chamber. Blot off any surplus fluid without disturbing the sample underneath the coverslip.

5. Transfer the slide to the microscope, and view a large square ruled by 3 lines using a 10x objective and 10x ocular. Count the total number of cells in 5 of the 9 major squares.
Count cells that overlap the top and left border of squares but not those overlapping bottom and right borders (Figure 1). This prevents counting overlapping cells twice. If the cell density is too high, the cell suspension should be diluted, noting the dilution factor.

6. Repeat the counting for the second chamber to give a total of 10 squares.

7. Add the number of cells counted in all 10 squares together to give the number of cells in 1 x 103 ml. Multiply by 1000 to give the number of cells
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