Viable cell count
- Check the chamber: it MUST be clean. If you can see some cells or other debris, clean it again with 70% ethanol and some paper and wait a few minutes until it dries.
- Prepare the cell suspension: wash and trypsinize the cells in the vessel. Check under the microscope approximately every minute for the cells to be detached and then inactivate the trypsin with complete medium at a 1:5 ratio. Make sure the cell suspension to be counted is well mixed. Transfer at least 10 ml into a 0.5 ml tube.
- Mix an equal volume of trypan bleu (attention: it’s toxic!) to the cell suspension in the 0.5 ml tube, load a 10 ul volume of the mix into the chamber and go to the microscope.
- Focus on the grid lines of the chamber and move to one set of 16 corner square as indicated by the diagram below.
- Count the cell number in this area of 16 squares, keeping separate the counts of the dead cells (blue stained) from the living ones (unstained).
Count only the cells within the square and any positioned on the right hand or top boundary line, to avoid counting twice some cells.
- Move the chamber to another set of 16 squares and carry on counting until all 4 sets of 16 squares are counted.
- Considering that the chamber is designed so that the number of cells in one set of 16 squares is equivalent to the number of cells multiplied by 104/ml, calculate the average of the living cells of the 4 counts from each set of 16 squares and than multiply the resulting number by 2, in order to adjust for the 1:2 dilution in trypan blue.
- Calculate the cell suspension viability by dividing the living cell number by the total (living+dead cells) cell number and multiplying by 100.