Klapperich Lab:Cell Counting Procedure

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How to count cells using Hemocytometer.


  • PBS
  • trypan blue soln.


  1. Aspirate media.
  2. Wash with 1X PBS.
  3. Aspirate PBS.
  4. Resuspend in media – make sure cells are completely resuspended, that they are not clumped together.
  5. Place coverslip on middle of hemocytometer.
  6. remove 500 ul of cells to microfuge tube.
  7. Add 50 ul of cell solution to 50 ul of trypan blue solution in a second microfuge tube.
  8. 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.
  9. Place hemocytometer on scope at 10X magification
  10. Locate center 5 x 5 grid.
  11. Move to higher power.
  12. 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.
  13. Count the cells in two other 1 mm2 areas (one of the squares surrounding the center square).
  14. Average the values for the final count.


  1. List troubleshooting tips here.
  2. You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
  3. Anecdotal observations that might be of use to others can also be posted here.

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Relevant papers and books

  1. 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. DOI:10.1073/pnas.78.11.6840 | PubMed ID:6947258 | HubMed [Goldbeter-PNAS-1981]
  2. JACOB F and MONOD J. Genetic regulatory mechanisms in the synthesis of proteins. J Mol Biol. 1961 Jun;3:318-56. DOI:10.1016/s0022-2836(61)80072-7 | PubMed ID:13718526 | HubMed [Jacob-JMB-1961]
  3. ISBN:0879697164 [Ptashne-Genetic-Switch]

All Medline abstracts: PubMed | HubMed


  • Who has experience with this protocol?