- No dilutions to obtain optimal counting density
- No tedious manual hand counting
- No strenuous peering into a microscope for extended periods of time
- Accurate -- cell aggregates do not affect counting accuracy
- Multiple samples can be counted at the same time
What is the PCV Cell Counting Kit and How does it work?
PCV Cell Counting Kits contain a cell counting tube that holds up to 1ml of cell suspension that can be centrifuged forcing the cells into a graduated 5µl capillary at the bottom of the tube. The volume occupied by the cell pellet after centrifugation is called the Packed Cell Volume (PCV). The Packed Cell Volume (PCV) can then be converted to a useful value such as PCV% or and actual cell number.
What is PCV% and how is it calculated?
The PCV% is the Packed Cell Volume divided by the cell suspension volume initially loaded in the cell counting tube. If 1ml of cell suspension is spun down to a PCV of 3µl, the PCV% would be calculated as follows:
PCV% = 3µl, 1000µl = 0.003 or 0.3%
The PCV% is a stand alone value that corresponds directly to the number of cells in culture. The PCV% can be used a measure of culture growth phase (as with bacteria), or as a benchmark for seeding or plating cultures for cell passage or experiments.
Can PCV% be used to calculate an actual cell number?
Yes, using either of two simple methods:
1) Parallel Count with a Hemacytometer
Perform a ONE-TIME, parallel count from the same culture using both a hemacytometer and the PCV Cell Counting Kit to obtain a conversion factor which can then be used for all future counts of that particular cell line.
Using 1ml of the same cell suspension for each; a cell count of 2,100,000 cells/ml is obtained with a hemacytometer, and the Packed Cell Volume (PCV) obtained using the PCV Cell Counting Kit is 3μl. The conversion factor would be calculated as follows:
Conversion Factor = 2,100,000 cells ¸ 3μl = 700,000 cells/PCVμl
The 700,000 cells/PCVμl conversion factor can then be used for all future cell counts of the particular cell line using the PCV Cell Counting Kit without the need of a hemacytometer.
2) Average Cell Diameter
If the average cell diameter (µm) of the particular cell line is known, then the diameter can be applied to a graph that's plots cells (millions) per PCVµl as a function of cell diameter(µm) in order to obtain cells/PCVµl
It is known that the average cell diameter of the cell line of interest is 17µm. From the graph it can be determined that there are approximately 380,000 cells/PCVµl. If 1ml of cell suspension is spun down to a PCV of 3µl, then there are 1,140,000 cells/ml (380,000 x 3).
What if the PCV is greater than 5μl?
Load a smaller volume of cell suspension, or if absolutely necessary, perform a dilution.
What if the PCV is too small to visualize accurately?
If possible, load more cell suspension.
Does Centrifugation Speed or Time Affect the Pellet Volume?
Yes, but the recommended speed and time (2400 x g/5,000 rpm for 1 minute) outlined in the PCV Cell Counting Kit protocol fall within ranges where the Packed Cell Volume (PCV) is constant and unaffected by centrifugation speed and time.
The packed cell volume of cells as a function of speed and centrifugation time.
A) Effect of centrifugatin speed:
Per tube of 300µl of CHO cells at 4.2 x 106 cells per ml were added.
B) Effect of centrifugation time:
Per tube of 400µl of CHO cells at 3.2 x 106 cells per ml were added (n=4).
Can living and dead cells be distinguished?
No. All cells regardless of health will be pelleted in the cell counting tube capillary.