8004708 Cellometer™ Ascend User Manual Rev A 25
Chapter 6. Viability Methods
This chapter describes how to choose a viability staining method based on the cell sample and selected assay type.
EVALUATING VIABILITY METHODS
When evaluating viability methods, it is critically important to use a single aliquot from the stock cell culture to
perform all testing. The cell sample should be evaluated for concentration on Cellometer Ascend prior to staining.
Note: Cell concentrations of 2.0 x 10
4
− 2.0 x 10
7
cells/mL can be analyzed on Cellometer Ascend, with a
concentration of 2.0 x 10
6
cells/mL being optimal.
If comparing the Trypan Blue and AO/PI viability methods, a portion of the sample should be stained with trypan
blue and another portion stained with AO/PI. Using samples from the same aliquot containing identical cell
concentrations will result in a more accurate comparison of staining methods.
Dilution or concentration of a cell sample may be required based on the initial concentration. It is recommended to
use cell culture media for dilution.
Using Trypan Blue Viability Method
Brightfield imaging and the Trypan Blue Viability can be used to determine the number, concentration and
percentage of live cells for cell lines and cultured primary cells. Brightfield imaging with trypan blue staining is not
recommended for samples containing debris, platelets or red blood cells. For accurately differentiating nucleated
cells, fluorescence is required.
PREPARING A CELL SAMPLE FOR TRYPAN BLUE VIABILITY DETERMINATION
Invert the tube containing cells ten times (10x) and pipette up and down 10x to generate a homogeneous cell
sample and reduce cell clumps. Do not shake or vortex the sample as this may damage cell membranes.
For viability measurement, stain cells by combining 20 μL of cell sample with 20 μL of a 0.2% trypan blue staining
solution (for a final concentration of 0.1% trypan blue). Gently mix by pipetting up and down 10x.
TRYPAN BLUE STAINING SOLUTION GUIDELINES
Use the following trypan blue staining solution guidelines when preparing cell samples for analysis.
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