How do you calculate cell count?

Cell counting is determined by using a hemocytometer and a specific formula to estimate the number of cells in a given sample. Here's a breakdown of how to calculate it, based on the provided references:

Calculating Cell Concentration

The primary step involves calculating the cell concentration, which is the number of cells per unit volume (typically per milliliter, or cells/ml). The formula is:

• Total cells/ml = (Total cells counted x Dilution factor x 10,000 cells/ml)/ Number of squares counted.

Here's how to use this formula:

1. Total cells counted: This is the sum of all the cells counted in the specified number of squares in the hemocytometer.
2. Dilution factor: This represents how much the original sample was diluted before counting. For example, if you mixed 10µl of sample with 10µl of diluent, the dilution factor would be 2.
3. 10,000 cells/ml: This is a constant that corrects for the volume of the hemocytometer grid.
4. Number of squares counted: This is the number of squares you counted within the hemocytometer.

Example:

Reference 2 provides an example:

• Total cells counted: 325 cells
• Dilution factor: 2
• Number of squares counted: 5

Applying the formula:

Total cells/ml = (325 cells x 2 x 10,000 cells/ml) / 5 = 1,300,000 cells/ml. This can also be expressed as 130 x 10⁴ cells/ml.

Calculating Total Cell Number in the Sample

Once you have the cell concentration, you can calculate the total number of cells in the original sample using the following formula:

• Total cells in sample = Total cells/ml x Total volume of sample (ml)

Using the result from the previous calculation and an example from Reference 3, if we had a total sample volume of 5 ml, the calculation would be:

Total cells in sample = 130 x 10⁴ cells/ml x 5 ml = 650 x 10⁴ cells

Practical Tips for Accurate Cell Counting

• Even Distribution: Ensure cells are evenly distributed in the sample before counting by mixing thoroughly.
• Proper Dilution: Dilute the sample appropriately to avoid counting too many or too few cells.
• Consistent Counting: Develop a consistent method for counting cells within the squares to avoid bias. For example, count cells touching the top or right lines only.
• Multiple Counts: Repeat cell counts multiple times and average the results for a more accurate measure.
• Viability Assessment: In many cases, it's essential to determine cell viability by staining dead cells before counting, usually using trypan blue which can exclude dead cells by passing through their broken membranes.
• Use Automated Counters: While manual counting is common, automated cell counters can significantly improve speed and accuracy in high-throughput experiments or routine analysis.

By understanding and implementing these steps, you can accurately calculate cell count in your samples.