*** Cell Viability Assay ***

• 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)