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LABORATORY PROCEDURES FOR ANIMAL & HUMAN CELL LINES

REFERENCE NO: AHC/1998/3/3.1/2.1


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

  1. Ensure the haemocytometer is clean and free form debris using 70% (v/v) ethanol.
  2. 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.
  3. Dilute the cell suspension 1:2 to 1:10 in medium to give an approximate cell concentration of 106/ml. Attached cell lines will need to be pre treated with trypsin to give a cell suspension.
  4. Dilute the cell suspension 1:2 with trypan blue (0.4% w/v).
  5. Load the chamber without over filling (approximately 20Ál). Allow the suspension to be drawn into the chamber by capillary action.
  6. Focus on the grid using the x10 objective.
  7. 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 1mm2. This area is then sub divided into 25 areas
  8. Count the number of cells (counting those stained and unstained separately) within the 1mm2 area.
  9. Calculate the viable cell count using the following equation.
  10. 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 = 104 conversion factor

  11. 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

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