Klapperich Lab:Cell Counting Procedure
How to count cells using Hemocytometer.
- trypan blue soln.
- Aspirate media.
- Wash with 1X PBS.
- Aspirate PBS.
- Resuspend in media – make sure cells are completely resuspended, that they are not clumped together.
- Place coverslip on middle of hemocytometer.
- remove 500 ul of cells to microfuge tube.
- Add 50 ul of cell solution to 50 ul of trypan blue solution in a second microfuge tube.
- Add 20 ul of the final solution to each side of the hemocytometer. by letting a drop of liquid get taken into the slid by capillary action.
- Place hemocytometer on scope at 10X magification
- Locate center 5 x 5 grid.
- Move to higher power.
- Count all of the viable cells in the 5 x 5 grid. It is easier to count at higher power. Dead cells will be dark blue.
- Count the cells in two other 1 mm2 areas (one of the squares surrounding the center square).
- Average the values for the final count.
- List troubleshooting tips here.
- You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
- Anecdotal observations that might be of use to others can also be posted here.
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Relevant papers and books
- Goldbeter A and Koshland DE Jr. An amplified sensitivity arising from covalent modification in biological systems. Proc Natl Acad Sci U S A. 1981 Nov;78(11):6840-4.
- JACOB F and MONOD J. Genetic regulatory mechanisms in the synthesis of proteins. J Mol Biol. 1961 Jun;3:318-56.
- Mark Ptashne. A genetic switch. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press, 2004. ISBN:0879697164
- Who has experience with this protocol?