Worthington Tissue Dissociation Guide

One of the simplest methods to approximate cell viability is the dye exclusion technique. This method utilizes an indicator dye to demonstrate cell membrane damage. Cells which absorb the dye become stained and are considered non-viable. Dyes such as trypan blue, erythrosin, and nigrosin are commonly used with trypan blue being the most common in preliminary cell isolation procedures.

This procedure can be performed along with the cell counting procedure but cell density may require adjustment in order to obtain approximately 10⁶ cells per milliliter.

Procedure

1. Mix 1 drop of trypan blue with one drop of the cell suspension and allow 1 - 2 minutes for absorption
2. Prepare hemocytometer and load chambers as described in "Cell Quantitation".
3. Count both the total number of cells and the number of stained (dark) cells.

Calculation

Percent Viability =
	Total Cells Counted - Stained Cells	x 100
	Total Cells Counted

Example

Total Cells / 1 mm² = 182
Stained Cells = 24

% Viability =	182 - 24	=	158	x 100
	182		182
		=	86.8% Viability

Note: Dye exclusion viability procedures tend to give high estimates of cell viability when compared to cell attachment or metabolic assays, but for optimization of cell isolation procedures trypan blue does provide a rapid estimate of dissociation performance in conjunction with yield quantitation.