How to Count Cells with a Hemocytometer: A Step-by-Step Guide
Cell counting is an important part of many different scientific experiments. In this step-by-step guide, we will show you how to use a hemocytometer to count cells and determine cell viability using trypan blue staining.
A Neubauer cell chamber, also called a hemocytometer, is a useful tool in cell counting and determining cell viability. The neubauer chamber consists of two parts: a counting chamber and a cover slip. The counting chamber is a square with evenly spaced lines on it, and the cover slip sits on top of it to keep the cells in place.
Processing cells for loading into the hemocytometer
The first step is to prepare the cell suspension. If you are counting cells from a liquid sample, such as blood or culture media, you will need to centrifuge the sample to pellet the cells. Once the cells are pelleted, you can resuspend them in a small volume of saline or water. If you are counting cells from a solid sample, such as a tissue slice or cultured cell monolayer, you can simply place the sample on the counting chamber.
- Add the cells to be counted into a tube. The number of cells should be enough so that individual cells can be seen and counted (at least 25-50 cells).
- Centrifuge the cell suspension for five minutes at 1000 RPM. This will pellet the cells and cause them to stick to the bottom of the tube.
- Carefully decant (pour off) the supernatant (liquid on top). Be careful not to disturb the cell pellet at the bottom of the tube.
- Add 500 μL of PBS to resuspend the cells. Swirl gently to mix.
A hemocytometer is a cell counting device that consists of a microscope slide with a grid etched onto the surface. The grid is used to count cells in a given area.
- Fill the Neubauer cell counting chamber with trypan blue solution. The trypan blue should come up to the line marked "0.04mm" on the Neubauer cell counting chamber.
- Next, add your cell sample to the Neubauer cell counting chamber. Be sure to add enough cells so that you can make accurate counts (at least 25-50 cells).
- Gently swirl the Neubauer cell counting chamber to mix the cell sample and the trypan blue solution.
Cell counting procedure
- Carefully place the Neubauer cell counting chamber on a microscope slide. Make sure that the grid lines in the Neubauer cell counting chamber are facing the microscope lens.
- Focus the microscope on the grid lines in the Neubauer cell counting chamber. You should be able to see individual cells within the grid.
- Count the number of cells in a given area (e.g., quadrant, sector, etc.).
- Repeat this process for the other areas in the Neubauer cell counting chamber.
To calculate the number of white blood cells (WBCs) per mL in your original sample:
- Calculate the WBC dilution factor (DF). DF = Number of cells in Neubauer cell counting chamber / number of WBCs counted per Neubauer cell counting chamber.
- Count the number of WBCs in each neubauer cell counting chamber. Multiply the number of WBCs counted per Neubauer cell counting chamber by the WBC dilution factor. This will give you the number of cells/mL in your original sample.
For estimating the number of red blood cells (RBCs) in a given volume:
- Count the total number of cells in a Neubauer cell counting chamber. This will include both white blood cells and red blood cells.
- Calculate the percentage of RBCs in your sample by dividing the number of RBCs counted by the total number of cells counted.
- Multiply the percentage of RBCs in your sample by the volume of your original sample. This will give you the estimated number of RBCs in your original sample.
Trypan blue cell viability assay
Trypan blue is a dye that is used to stain live cells. When the dye binds to a cell, it causes the cell to take on a blue color. This makes it easy to distinguish between live and dead cells. Live cells will take up trypan blue, while dead cells will not.
- To calculate cell viability, you will need to know the total number of cells counted and the number of live cells (stained with trypan blue).
- The formula for cell viability is: % Cell Viability = (Number of Live Cells/Total Number of Cells) x 100.
- For example, if you counted 100 cells and there were 30 live cells, the cell viability would be: (30/100) x 100 = 30%.
- To calculate the percentage of dead cells, you would use the same formula, but substitute "Number of Dead Cells" for "Number of Live Cells".
Tips for improving efficiency of cell counting in a hemocytometer
- Count cells in multiple areas of the Neubauer cell counting chamber to get a more accurate estimate of the number of cells in your sample.
- Try to use the same area of the Neubauer cell counting chamber for all of your counts. This will help you to be more accurate and consistent in your measurements.
- When using the neubauer cell counting chamber to count cells in a sample it is important to use a microscope with at least 40x magnification. This will allow you to see individual cells within the grid.
- To stain the cells, mix a small volume of trypan blue with your sample and let it sit for at least five minutes. Be careful not to disturb the cells on the cover slip.
- Use a microscope with a digital camera to take pictures of the neubauer cell counting chamber. This will allow you to save time by not having to count cells manually.
- Use a cell counting software program to automate the cell counting process. This can be a real time saver, especially if you are counting a large number of cells.
The Neubauer cell counting chamber is a handy tool that can be used to count cells and determine cell viability. We hope that this article was helpful and now you feel confident using a hemocytometer! Have any tips of your own? Share them with us.
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