Cell viability test by Trypan Blue

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BACKGROUND

Trypan Blue dye is used to differentiate viable cells present in a cell suspension from dead cells based on the concept that viable cells have intact cell membranes that exclude Trypan blue, whereas non-viable cells cannot. A viable cell will have a clear cytoplasm whereas a nonviable cell will have a blue cytoplasm.

Aim: Aim of this experiment is to test viability of cell by trypan blue.

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REQUIREMENTS

Sample:        Cell suspension

Apparatus:    Haemocytometer

          Coverslip

          Pipettes

          Tips

Instrument:    Light microscope

Chemicals:    0.4% Trypan blue solution

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PROCEDURE

Make a cell suspension from the cells cultured in the plate. To a 50µl cell suspension in a cryovial, add 50 µl of 0.4% Trypan blue solution (ratio 1:1) mix it gently. Keep the cryovial at room temperature for less than 3 min. Place the coverslip on the haemocytometer, fill one side of haemocytometer with cell suspension by placing the tip of the pipette at the notch. Typically, each side will take 10 to 20µl. check in the light microscope, haemocytometer has multiple squares in each side. Count all the cells in each large square (see Fig. 1) in each corner of the haemocytometer. In each large square count cells that are on the border lines on two sides only. Keep track of the number of blue cells separately as well as part of the complete number of cells. Blue cells are the nonviable cells.¹

Calculation

Calculate the percentage of viable cells by dividing the number of viable cells.²

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CONCLUSION

Using the formula, we can thus calculate the percentage of viable cells in a given cell suspension.

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REFERENCES

1. Kumar, A., Mishra, H.K.,Dwivedi, P., Subramaniam (2015). Secreted trophic factors of Human umbilical cord stromal cells induce differentiation and neurite extension through PI3K and independent of cAMP pathway. Annals of neuroscience. 2015;22(2):97-106.
   
2. Social and scientific systems inc. NIEHS, NIH.