Biology
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Summary
Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. In this video, we demonstrate how cells are counted using a hemacytometer.
Cite this Article
Copy Citation | Download CitationsRicardo, R., Phelan, K. Counting and Determining the Viability of Cultured Cells. J. Vis. Exp. (16), e752, doi:10.3791/752 (2008).
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Abstract
Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. A hemacytometer is a thick glass slide with a central area designed as a counting chamber. Cell suspension is applied to a defined area and counted so cell density can be calculated.Protocol
The complete text protocol for this experimental approach is available in Current Protocols in Cell Biology.
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Disclosures
The authors have nothing to disclose.
Comments
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I think it would be helpful if you provided links to related protocols (such as for passaging cells, as referenced in the protocol). For example, have a "Related protocols" section with direct links to them.
If I take ²00microliter.spin it down and resuspended in ²0 microliter then should I divide result by 10 ? Is that right?
If you are talking about the concentration of cells in suspension, then no, you multiply by ten. When you decrease the volume by a factor of ten, then you are concentrating cells and should increase the concentration by a factor of ten.
I thought it's the opposite.Let's say if you dilute the cells 10x won't you multiply by 10 and it should be the oppsite in concentrating?
Divide by 10 since you have made the solution more concentrated than it actually is.
So let's make sure we are talking about the concentration of cells, which is what you get after counting them. concentration of your cells is defined as equal to the # cells/ unit volume. so, if you spin down your cells, which were at ²00 microliters, and then resuspend in ²0 (thereby decreasing the volume by a factor of 10) you can multiply the concentration by a factor of 10 (because they are inversely proportional as the definition states). Or if you feel like being fancy multiply ²00 microliters, which is in the denominator by 0.1 (because ²0 is a 10th of ²00). when you dilute cells you are doing the opposite, increasing the volume that contains a fixed # of cells will lower the concentration, because in your example you are multipling the volume (in the denominator) by a factor of 10. divide the concentration by 10.
Overflowing the center posts when you are loading the cell suspension into the hemacytometer results in liquid flowing over and cells staying behind, so that the count is artificially raised. The cell suspension loaded onto the stage area (usually about 10-15 uL) should only be allowed to flow (in one swoop, not in jerks as shown) to cover the counting surface, then stopped so that liquid dŒs not overflow into the side wells. This technique as shown is less than desirable.
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I agree with C. Kolar in his/her comment about not letting the liquid overflow; although it requires some manual sensitivity for not pushing too hard the micropipette plunger, it is something everybody should learn how to do. I would like to make some suggestions to improve the work. First, I would invite authors to consider and discuss subjectivity in this type of cell counting; sometimes it is very subjective to decide how blue a cell should be to be considered dead;. In second place, letting the cells 5 minutes into the trypan blue solution might lead to cell death (and therefore decreased cell viability) if not properly stored; so this time should be standardize for every cell type. The authors also should consider explaining the remaining cell counting area, not just the central part. Some people consider the remaining 4 zones of 1 square mm for cell counting and use all of them to get an average instead of one single value from the central zone. When calculating the cell concentration, the authors do not explain why the "10.000" appears in the formula (just the product of 1 square millimeter (area) and 0.1 mm (height of the chamber)), and what is considered as dilution...I believe an example would be very helpful to explain this aspect and is missing from the presentation.