BACKGROUND
Haemocytometer
In this experiment, we will passage (subculture) cells and count it on the haemocytometer. Adherent cells needs to be subculture after getting confluency (more than 70%) in the plates without it cells will remain unhealthy and sensitive for contaminations. To detach the cells from the plate and harvest in another plate we need to trypsinize and harvest in another plate.1
REQUIREMENTS
Sample:
Animal cells in confluent stage in a plate.
Apparatus:
- Sterile petri dishes
- Serological pipettes
- Centrifuge tubes – 15 ml
- Centrifuge with rotor
Chemicals:
- Cell culture media
- 0.5% trypsin-EDTA solution 10X
- 1X HBSS
PROCEDURE
| Content of cell culture media | Composition |
| DMEM | 500 ml |
| Serum | 50 ml |
| Antibiotics/Antimycotics | 500 µl |
Take out the confluent cell plate and wash with 1X HBSS (5-10 ml/60 mm petri dish). Add trypsin solution in the petri dish (5-8 ml/60 mm petri dish), keep it in the incubator for 5 min and check all the cells were detached, add 3-5 ml cell culture media, transfer the solution in the 15 ml centrifuge tube and mix it gently to make sure that cell are completely dissociated from each other.
Further centrifuge the tube at 300x g for 3-5 min discard the supernatant and add 5 ml cell culture media. Take 20 µl of the solution, and place it on the haemocytometer and count the cells in the five large square and take the average of it and multiply with 104. That is the total no. of cells/ml.2
Calculation
Total no. of cells in five large square- 250
Average no. of cells in a large square- 50
Concentration of cells- 50 x 104
CONCLUSION
We concluded the trypsinization and cell counting of the cells with the help of haemocytometer.
REFERENCES
- Grange C, Bussolati B, Bruno S, Fonsato V, Sapino A, Camussi G.. Isolation and characterization of human breast tumor-derived endothelial cells. Oncol Rep. 2006;15(12):381-6.
- Kumar A, Mishra HK, Dwivedi P, Subramaniam JR (2015). Secreted trophic factors of Human umbilical cord stromal cells induce differentiation and neurite extension through PI3K and independent of cAMP pathway. Ann Neurosci. 2015;22(2):97-106.
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