Quantification of Cell Number
Note: In order to obtain a cell count with the PCV Cell Counting Kit, it is required that a ONE-TIME parallel count be performed with a hemacytometer or other method to determine the factor necessary to convert Packed Cell Volume (PCV) to an actual cell number. This parallel count must be performed only ONCE with a particular cell line to establish the proper conversion factor, and is not required for all subsequent counts of that cell line.
Parallel Count Method
- Mix cell suspension and add up to 1ml to the counting tube.
- Centrifuge at 5000 rpm/ 2400 x g for 1 minute.
TIP: Either swing-out or fixed angle microcentrifuge rotors are acceptable for centrifugation and accurate determination of Packed Cell Volume (PCV). However, a swing-out rotor is preferred as a level cell pellet is achieved which is makes it easier to accurately visualize the Packed Cell Volume (PCV) in the tube capillary. If a 1.5/2.0ml swing-out rotor is not available, the cell counting tube (with lid) can be dropped into a 15ml conical centrifuge tube and placed in a swing-out rotor for 15ml tubes. The 15ml tubes included with these kits have the tolerances tobe spun at the speeds necessary for complete cell pelleting.
- Read the Packed Cell Volume (PCV) in μl from the cell counting tube graduated capillary. If the Packed Cell Volume (PCV) is greater than 5μl, load less cell suspension and spin again.
- Perform a cell count of the cell suspension using a hemacytometer or other method.
- Determine the conversion factor to be used for all future counts of the particular cell line with the PCV Cell Counting Kit.
If 1ml of cell suspension yields a Packed Cell Volume (PCV) of 3μl and the cell density as determined with the hemacytometer is 2,100,000 cells/ml, then the conversion factor would be calculated as follows:
Conversion Factor = 2,100,000 cells ¸ 3μl = 700,000 cells/μl
For all future counts with this cell line, 1μl of Packed Cell Volume (PCV) would be equivalent to 700,000 cells. A Packed Cell Volume (PCV) of 2μl would be equivalent to 1,400,000 cells, etc.
Cell Diameter Method
If the average cell diameter of the cell line of interest is known, the cell number can be determined using the graph below. Obtain the Packed Cell Volume (PCV) using steps 1-3 of the Parallel Count Method detailed previously.
If the average cell diameter of the cell line of interest is 17μm, then it can be extrapolated from the graph that there are approximately 380,000 cells per μl of Packed Cell Volume (PCV). If the PCV of a sample of that cell type was 2μl, there would be 760,000 cells in the volume of cell suspension initially loaded into the cell counting tube.
Calculation of PCV%
If an exact cell number is not required, the PCV% can be a useful value as a stand-alone measure of cell density or culture growth phase.
- Determine the Packed Cell Volume (PCV) using steps 1-3 of the Parallel Count Method detailed previously.
- Calculate PCV%
The PCV% is calculated by dividing the Packed Cell Volume (PCV) by the volume of cell suspension initially loaded into the cell counting tube.
If the PCV from 500μl of cell suspension was 2.5μl, the PCV% would be calculated as follows:
PCV% = 2.5μl ¸ 500μl = 0.005 or 0.5%
The PCV% value can be used as a reference for measuring culture growth, or for passaging or plating cells.