My intention is to plate the cells in 24 well plates followed by transfection. Although the flasks are 100% confluent, the cell count per square that I'm getting using the hemocytometer is not matching to the exact cell density of the flask before trypsinization.
Usually, I collect cells in 2ml of L-15 medium suspension after trypsin treatment. Then I prepare 20 ul of loading suspension (10 ul of cells and 10 ul of trypan blue dye). To load the cells into the hemocytometer I use 10 ul from the loading suspension. But when I count the cells using a hemocytometer hardly I'm getting less than 30 cells per square. I'm experiencing this issue only in the cell line that I developed. To cross-check this I have used another 100% confluent cell line (EPC) for counting where I'm getting more than 200 cells per square.
Could someone assist me with this? Thanks in advance.
Is it possible that after trypsinization, your cell line is either not yet a single cell suspenstion, or cell rupture has formed clumps of debris? Either one would prevent optimal spreading across the hemocytometer. What does the suspension look like before being loaded onto the hemocytometer?
Prof. Ellen A G Chernoff Thanks for the response. I'm working on an adherent cell line and the suspension is clear without any clumpings after the trypsin treatment. The spreading is also good on the hemocytometer.
Do you ever calculate the average cell density of your targeted cells during daily maintenance? It suppose to be similar. Particularly adherent cell lines, which cell density is closely related to the surface area of your container. Please refer to the following website.
https://www.thermofisher.com/tw/zt/home/references/gibco-cell-culture-basics/cell-culture-protocols/cell-culture-useful-numbers.html
Thanks, Prof. Shian-Ren Lin. The density of the cells at confluency is higher than 1* 106 (approx). Routinely, I trypsinize 85-100% confluent flask and followed by seeding the cells in a 25cm2 flask at a split ratio of 1:2. The problem I encounter is only with the cell count using a hemocytometer before plating them.
You mean that every time you harvest cell from 85%~100% confluent 25T, you can collect 1 * 10^6 cells, right? This information tell me that the ideal cultural density of your desired cell is 4 * 10^6 cells/cm^2. The surface area of 24-well plate is 1.9 cm^2, which means you can collect 7.6 * 10^4 cells from one well of 24-well plate. If there is a case, you count cells from hemocytometer less than 10 is reasonable.
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