How to Measure Cell Viability | eHow.com http://www.ehow.com/print/how_6114019_measure-cell-viability.

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By Sarah Quinlan, eHow Contributor

In most biological experiments involving cell culture, a

critical step includes knowing which cells are alive and
which are dead in your sample. Dye exclusion is the most
popular method of measuring cell viability. Dye exclusion is
based on the theory that live cells contain intact membranes
and dead cells do not, thus dead cells absorb the dye into the
cytoplasm. There are different dyes and protocols and the
methods you choose should be based on cost, sensitivity,
ease and length of procedure. Trypan blue is a common dye
used to measure cell viability because the procedure can be
done in five to 10 minutes and costs only the amount of the
dye reagent.

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How to Measure Cell Viability | eHow.com http://www.ehow.com/print/how_6114019_measure-cell-viability.html

Difficulty: Moderate

things you'll need:

0.4% trypan blue reagent
Hemacytometer
Cover slip
Micropipette
Pipette tips
Phosphate buffered saline (PBS)
Light microscope
Obtain a pellet of the cells you're measuring.

Resuspend the cell pellet in PBS to get approximately 5 x

105 cells/ml. PBS is used because viability measurements
are more accurate when cells are in a serum-free
environment; serum proteins may also stain and give you
misleading results.

Add equal parts of cell suspension in PBS to equal parts

of 0.4% trypan blue dye to obtain a 1 to 2 dilution
(example: 100 ul of cells to 100 ul of trypan blue) and
mix by pipetting up and down.

Incubate mixture for less than three minutes at room

temperature. If cells are counted after approximately five
minutes, viability will be inaccurate due to cell death.

With the cover slip already in place, fill each side of a

hemacytometer counter with the cell suspension.
Typically, each side will take 10 to 20 ul.

Place the hemacytometer on the stage of a light

microscope and focus onto the cells.

Each side of the hemacytometer contains multiple

squares. Count the total number of cells in one square of
the hemacytometer. Then count the number of non-viable
(blue) and viable (clear) cells separately, in the same
square.

Calculate the percentage of viable cells in the square by

dividing the number of viable cells by the number of total
cells and multiplying by 100. Do this for multiple squares
on the hemacytometer to obtain an average viability
measurement.

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Counting dead cells can be subjective, depending on what the technician considers a
blue cell (the intensity of the blue color may vary depending on how much was
absorbed into the cytoplasm). Therefore, it is wise to do a viability measurement with at
least two separate samples and obtain an average. Alternatively, the technician could get
another person to also perform the count and compare results.

Use personal protective equipment when performing this assay, such as gloves and a lab
coat. According to the Material Safety Data Sheet (MSDS), trypan blue may cause
cancer, so practice appropriate laboratory safety methods.

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