L-Cell dilution series and hemocytometer usage

After a two-week vacation, it was refreshing to get back to my internship at Applied Biophysics. It was great to get back to the lab, creating a new co-culturing experiment and learning learning how to count cells using a Hemocytometer.

Before I delve into the details of my internship on Friday, I'd like to write a quick happy birthday to the amazing Dr. Ivar Giaever who celebrated his 85th birthday on Saturday! An esteemed physicist, Dr. Ivar Giaever is a professor emeritus at RPI, a professor-at-large at the University of Oslo, and president of Applied Biophysics. And did I mention he won the 1973 Nobel Prize in Physics for his studies in tunneling phenomena in superconductors? I love hearing stories about Dr. Giaever, from his stories of his travels to across the world, to his take on global climate change. Happy birthday!

Back to the science! I am now working on another dilution series that has the same format as my past 96w20idf experiments with MDCK and BSC-1 cells, but instead of MDCK, I am using L-cells (mouse endothelial cells). The object of this experiment is to compare the behaviors of our previous cell combinations of MDCK and BSC-1 (epithelial/epithelial) to a mixture of BSC-1 and L-Cells (epithelial/fibroblast). Here is the layout of this 96w20idf dilution series:

Cell Counting using Hemocytometer:

Once I finished setting up my dilution series experiment, I learned how to use a hemocytometer to manually count cells. The idea behind this device is to take a small, manageable amount sample of cell mixture, and use it to make an estimate of how many cells are present in a larger volume of that same mixture. Hemocytometers are commonly used to count blood cells (hence the name hemo (meaning blood) cyto (meaning cell) and meter (meaning count)). Here is what a hemocytometer looks like:

To use the hemocytometer, one pipettes a small drop of cells suspended in medium into the tiny v-shaped notch as seen in the image above. There is a cover slide that sits on top of the raised part of the hemocytometer, which the cell solution slips under and fills. Capillary action draws the liquid into the space between the cover slip and the glass surface, creating a uniform layer of liquid .1mm in depth. There is a microscopic, laser-etched grid on both sides of the hemocytometer, which looks like this:

Using the applicable objective, I magnified one of the 4x4 squares, and counted the number of cells within this square. With cells that are on the edge of the square, only count those that touch the top and left sides of the square (it's just common practice). To calculate the cell concentration per ml, use this simple formula:

I counted up 5 of the 4x4 squares for both a sample of L-cells and a sample of BSC-1 cells which were left over from my dilution series experiment. I counted up 32 L cells and 34 BSC-1 cells, two values that are relatively similar. This similarity is good because that means the cell solutions used in my dilution series will be relatively reliable. 

We did not perform this in my experiment, but one can use a hemocytometer to calculate the cell viability count by staining cells with Trypan Blue. Trypan Blue is a "vital stain" that colors dead cells  blue due to the incorporation of color into the proteins of dead cells, and leaves live cells colorless.