In this article, we will provide a detailed protocol for obtaining a viable cell count from suspension cells using a hemocytometer. This protocol is essential for accurately determining cell numbers and ensuring consistency between experiments. By following these steps, you can obtain reliable and reproducible results.
Preparing the Hemocytometer
Before starting the cell counting process, it is important to prepare the hemocytometer properly. If you are using a glass hemocytometer and coverslip, clean them with alcohol before use. Moisten the coverslip with water and affix it to the hemocytometer. The presence of Newton's refraction rings under the coverslip indicates proper adhesion. If you are using a disposable hemocytometer, simply remove it from the packet before use.
Preparing the Cell Suspension
To ensure accurate results, it is crucial to follow aseptic technique to prevent contamination of cell cultures and reagents by microorganisms. We recommend watching our aseptic technique video protocol, which demonstrates how to sterilize work areas and use appropriate sterile handling techniques, personal protective equipment, and good hygiene.
Once you have prepared your work area, gently swirl the flask containing the cell suspension to ensure the cells are evenly distributed. Before the cells have a chance to settle, take out 0.5 mL of the cell suspension using a sterile pipette and transfer it to an Eppendorf tube.
Next, take 100 µL of the cell suspension and add 400 µL of 0.4% Trypan Blue (final concentration 0.32%). Mix the solution gently. Trypan Blue is a vital stain that helps distinguish between live and dead cells during the counting process.
Counting the Cells
To begin the cell counting process, take 100 µL of the Trypan Blue-treated cell suspension and apply it to the hemocytometer. If you are using a glass hemocytometer, very gently fill both chambers underneath the coverslip, allowing the cell suspension to be drawn out by capillary action. If you are using a disposable hemocytometer, pipette the cell suspension into the well of the counting chamber, allowing capillary action to draw it inside. Take care not to overfill the counting chamber.
Using a microscope, focus on the grid lines of the hemocytometer with a 10X objective. Using a hand tally counter, count the live, unstained cells (cells that do not take up Trypan Blue) in one set of 16 squares. It is important to follow a systematic counting method, where cells are only counted when they are set within a square or on the right-hand or bottom boundary line. Repeat this process for all four sets of 16 squares on the hemocytometer.
Calculating Viability and Cell Concentration
To calculate the number of viable cells per milliliter, take the average cell count from each of the sets of 16 squares. Multiply this average by 10,000 (104) and then multiply by 5 to correct for the 1:5 dilution from the Trypan Blue addition. The final value represents the number of viable cells per milliliter in the original cell suspension.
For example, if the cell counts for each of the 16 squares were 50, 40, 45, and 52, the average cell count would be:
(50 + 40 + 45 + 52) ÷ 4 = 46.75
46.75 x 10,000 (104) = 467,500
467,500 x 5 = 2,337,500 live cells per milliliter in the original cell suspension.
If you have recorded both live and dead cell counts for each set of 16 squares, you can calculate the percentage viability by dividing the live cell count by the total cell count. For example, if the live cell count is 2,337,500 cells/mL and the dead cell count is 50,000 cells/mL, the viability would be:
2,337,500 ÷ (2,337,500 + 50,000) = 97.9% viability
Conclusion
Obtaining a viable cell count from suspension cells using a hemocytometer is a crucial step in cell culture research. By following the protocol outlined in this article, you can ensure accurate and reproducible results. Remember to maintain aseptic technique throughout the process and carefully count the cells using a systematic approach. Calculating viability and cell concentration will provide valuable information for your experiments and help you make informed decisions in your research.
Keywords: viable cell count, suspension cells, hemocytometer, aseptic technique, Trypan Blue, cell concentration, viability.
