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| 发布时间：2006-09-27|
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Dye exclusion
- 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)
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Crystal Violet 