Viability assays are used to detect potentially damaging processes such as primary isolation, cell separation, or the percentage of living cells after freeze-thawing. Most viability assays are based on the disruption of membrane integrity, which can be measured by the uptake of a dye that normally does not enter the cell (e.g., Taipan Blue, Phaeocyanin, or Naphthalene Black), or that is normally taken up and stored by the living cell, and released at that time (e.g., Diacetyl Fluorescein or Neutral Red). However, this effect is transient and does not always predict eventual survival. In addition, dye rejection is always an overestimation of cell viability; for example, 90% of cells preserved in liquid nitrogen may reject trypan blue after melting, but only 60% of the cells prove to be able to adhere to the wall after 24h. Source: Animal Cell Culture: A Guide to Basic Techniques, Fifth Edition.
Operation method
Scheme 22.1 Dye rejection assay for cell viability assay
Principle
Naphthalene black, Tepan blue, and many other dyes [KahenbachetaL1958] are not permeable to living cells. The cell suspension was mixed with the dye and examined under a low-power microscope.
Materials and Instruments
Trypsin Move Materials For more product details, please visit Aladdin Scientific website.
Pasteur pipette Microscope Hand-held counter Survival dye Hematocrit plate
Aseptic
Subject cells, such as trypsin-digested adherent cells, thaw-frozen cells, or primary discrete cells
Suitable growth solution 20 ml
0.25% Trypsin 5ml
D-PBSA 10ml
Non-sterile
Hematocrit plate
Viability dye 1ml (e.g. 0.4% Taipan Blue or 1% Naphthalene Black prepared with D-PBSA or HBSS)
Pasteur pipette
Microscope
Hand Counter
Operating Procedure
1. Prepare a highly concentrated cell suspension ( ~1X106 cells/ml) by digestion with trypsin or centrifugation and resuspension.
2. Take a clean blood cell counting plate and set the coverslip between it in the appropriate position (see Scheme 21.1 and Figure 21.1).
3 . Mix one drop of cell suspension with one drop of (Tepan blue) or four drops of (naphthalene black) dye.
4 . Add to the counting chamber of a blood cell counting plate (see Scheme 21.1).
5 . Allow to stand for 1 to 2 min (but do not leave for long or the live cells will be damaged and absorb the dye).
6 . Place the counting plate under a microscope, select a 10X objective and focus using the gridlines (Fig. 21.1).
7 . Count the total number of cells as well as the number of colored cells.
8 . Wash the blood cell counting plate and place it in the cassette. 
