TITLE: CELL ENUMERATION BY VITAL DYE EXCLUSION
INTRODUCTION
This procedure describes the enumeration of cells using a haemocytometer. The number within a defined volume is counted and the cell concentration derived from the count.
PROCEDURE
- Ensure the haemocytometer is clean and free form debris using 70% (v/v) ethanol.
- Wet the edges of the coverslip and place it on the chamber. Move in small circular motions until the coverslip sticks and newtons rings are visible.
- Dilute the cell suspension 1:2 to 1:10 in medium to give an approximate cell concentration of 10^{6}/ml. Attached cell lines will need to be pre treated with trypsin to give a cell suspension.
- Dilute the cell suspension 1:2 with trypan blue (0.4% w/v).
- Load the chamber without over filling (approximately 20µl). Allow the suspension to be drawn into the chamber by capillary action.
- Focus on the grid using the x10 objective.
- Move the slide so that the central area if the grid comes into view. This is the largest area and is bounded by three parallel lines. This area is 1mm^{2}. This area is then sub divided into 25 areas
- Count the number of cells (counting those stained and unstained separately) within the 1mm^{2} area.
- Calculate the viable cell count using the following equation.
a x d x e x f = cells per ml
where a = the number of viable cells (unstained)
d = the dilution factor in trypan blue
e = the dilution factor in medium
f = 10^{4} conversion factor
- Calculate the % viability using the following equation
100 x number of viable cells
total number of cells (stained plus unstained)
Guidelines prepared for CABRI by CERDIC, DSMZ, ECACC, INRC, November 1998
Page layout by CERDIC
Copyright CABRI, 1998