a cell suspension is mixed with trypan blue and examined by low-power microscopy
Materials
cells
PBS
M3
hemocytometer
0.4 % trypan blue in PBS
micropipet
microscope
Protocol
prepare cell suspension at a high concentration (ca 106 cells/ml)
take a clean hemocytometer slide and fix the coverslip in place
clean the surface of the slide with 70 % EtOH, taking care not to scratch the semi-silvered surface
clean the coverslip, press it down over the grooves and semi-silvered counting area
mix one drop of cell suspension with one drop of trypan blue
collect about 20 ul into the tip of a micropipet
transfer immediately to the edge of the coverslip, and let the suspension run into the counting chamber, the fluid should run to the edges of the grooves only
leave 1 - 2 min (do not leave longer)
place on microscope under a 10X objective and focus on grid lines in chamber
move the slide so that the largest area you can see is bounded by three parallel lines (1-mm2)
count the cells lying within this area.
count cells that lie on the top and left-hand lines of each square but not those on the bottom or right-hand lines
hundreds of cells per 1-mm2 area is ok
if there are less than 100 cells, count one or more additional squares (each surrounded by three parallel lines) surrounding the central square
count the number of stained cells and the total number of cells
wash hemocytometer
Dye exclusion viability tends to overestimate viability
Most viability tests rely on a breakdown in membrane integrity determined by the uptake of a dye to which the cell is normally impermeable (e.g., trypan blue)
