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        Cell Viability Assay

        互联网

        1575

         

        • 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)
        (责任编辑:大汉昆仑王)
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