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