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 (ca106 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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