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  • Writer's pictureNanoEntek

Ch.4-1 Trypan blue for brightfield

Updated: Jun 2, 2023

Having your cells counted with automated cell counters can be hassle-free. Yet, deciding which cell counter to use could be confusing and difficult at first.

In broad aspect, there are two different types of automated cell counter – brightfield (BF)-based and fluorescent (FL)-based in image-based cell counter. Depending on the cell counter type, staining solution or dye required may vary as well.

In the following two posts, a brief description about different types of staining solution/dye will be introduced.

[For brightfield-based cell counter]

1. Trypan blue (TB)

  • Definition:

Trypan blue (TB) is a dye standardly used for cell counting. It is a membrane-impermeable dye so that it only stains the dead cells.

  • Mechanism :

TB only stains the dead (non-viable) cells by entering through broken cell membrane, which protects the inner part of a cell. Upon entering, negatively charged TB interacts with the positively-charged nucleus of cell. With aid of component such as lysis buffer (a cell lysis reagent that breaks cell membrane), TB is then able to dye both live (viable) and dead cells for viability.

Fig.1 Staining method of trypan blue (TB) or trypan blue exclusion method

  • What to consider:

Although TB is an inexpensive and commonly used dye, it could penetrate through live cells after certain period of time and becomes toxic to cells. Thus, it could affect the cell conditino and viability result. What to consider it that TB is known to be harmful not only to cells but to humans as well. TB is identified as a carcinogen which may cause cancer in human after a long period of exposure.

An automated cell counter

Analysis time

< 20 seconds/test


Trypan blue

Cell type

Cell line (clumped cell,

Measuring range

1x10E4 to 1x10E7 cell/mL

The world's fastest automated cell counter

Analysis time

<1 seconds (manual focus)


Trypan blue

Cell type

Cell line (clumped cell, single cell)

Measuring range

1x10E4 to 2x10E7 cell/mL

A high-throughput automated cell counter

Analysis time

3 minutes/48 tests


Trypan blue

Cell size range

Detectable: 1-85 μm

Optimal: 5 - 80 μm

Measuring range

Detectable: 1x10E4 to 2x10E7 cells/mL

Optimal: 1x10E5 to 1x10E7 cells/mL


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