35 Viable Cell Enumeration
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- Objective micrometer
- Pasteur pipettes
Procedure
1. When counting cells in a suspension, shake the culture vessel to suspend the cells homogeneously. Leave the cell suspension for several
minutes to settle the cells. In the case of substrate-dependent cells, the
culture is subjected to the following procedures without any prior treatment.
2. Place the culture vessels on the stage of a microscope and, select five
fields at random.
3. Count the number of cells in a preset number of squares.
4. From the data obtained in step 3, the average number of cells in a unit
area can be calculated.
5. Measure the area of the bottom of the culture vessels.
Results
Cell density can be calculated from the number of cells (n) per 1 mm 2 ,
the area of the bottom of the vessel (w; mm 2 ), and the volume of the
culture medium (v; ml) using the following equation:
Cell density (cells/ml) = (n x w)/v
Subprotocol 3
MTT Method
Materials
- Multiwell plates
- Multiplate centrifuge
- Multiplate mixer
- Microplate reader
- MTT solution (5 mg MTT/1.0 ml distilled water)
Procedure
1. Culture cells in a multiwell plate.
2. Add 10 III sterile MTT solution to each well.
3. Culture the cells for a further 4-6 h.
4. Centrifuge the cells at 150 gfor 5 min,and discard the culture medium.
5. Add 100 jll dimethylsulfoxide to each well in order to extract formazan
formed in the cells.
6. Stir the solution using a multi plate mixer.
7. Read the absorbance of the solution at 570 nm with microplate reader.
Equipment
Solutions