Dear colleagues,
I am currently using HCT-116 adherent cells for cell culture in 96-well plates and have some questions regarding cell counting. I have tried various methods and found the most suitable to be adding 30 µL of trypsin-EDTA to assist in cell detachment, followed by adding 70 µL of medium to neutralize the reaction. I then use a pipette to repeatedly pipette up and down to help detach the cells before taking samples for counting.
Since I only need to count cells and not retrieve them, is it reasonable to leave the trypsin in the plate? Also, if the cell density is low, could I use a smaller volume of trypsin and medium to resuspend the cells?
Hello Mars Yang,
For a 96-well plate, cell counting with hemocytometer would be laborious and prone to variable results.
Moreover, there are certain disadvantages when you are trying to compare cell counting using Trypan Blue exclusion assay with CCK-8 assay.
Trypan blue stains dead cells in shades ranging from light blue to black, depending on the overall viability of the cell culture. When using a bright-field microscope, cells that are very lightly stained with trypan blue can be hard to differentiate from unstained cells, and thus hard to identify. Also, Trypan blue starts to become toxic to cells with passage of time. Even viable cells are eventually stained with Trypan blue, as when the dye permeates their cell membrane, they die over time. Therefore, the viability analysis can change over time, resulting in underestimation of the viability of a cell population.
So, I feel you should use an assay similar to CCK-8 assay like the AlamarBlue assay. The AlamarBlue cell viability assay is used to quantify cellular metabolic activity and in turn determine the concentration of viable cells in a given sample.
The AlamarBlue dye in its oxidized form is blue in color and non-fluorescent. In
AlamarBlue assay, the growing cells cause a chemical reduction of the AlamarBlue dye from non-fluorescent blue to fluorescent red. The continued growth of viable cells maintain a reducing environment (fluorescent, red) and inhibition of growth maintains an oxidized environment (non-fluorescent, blue), which can be detected using a fluorescence or absorbance detector. The protocol is simple. You need the AlamarBlue reagent and a plate reader.
You may follow the below protocol.
1. Warm-up cell culture media to 37°C.
2. Prepare the working solution of AlamarBlue reagent by diluting it with warm cell culture media 1:10.
3. Replace cell culture media with working solution of AlamarBlue from the previous step. Incubate in cell incubator at 37°C for 1-4 hours. Optimization of the incubation time might be needed.
4. After incubation, collect media with AlamarBlue from each well and pipette 100 μl/well to an optical 96-well plate for plate reader measurement.
5. For fluorescence measurement, the AB excitation range is 540–570 nm and emission range is 580–610 nm. Spectrophotometrically, the AB absorbance can be read at 570 nm, using 600 nm as a reference wavelength.
6. To calculate the % reduction of alamarBlue reagent, you may refer to the link below.
https://www.bio-rad-antibodies.com/measuring-cytotoxicity-proliferation-spectrophotometry-fluorescence-alamarblue.html
Best.
Hey,
Normally, we wouldn't do cell counting for this test, especially in a 96 well plate formate. If you want to know the number of cells, I suggest using the cell titer glo kit. With this kit, you could try titrating your cells with different densities to create a standard curve for analyzing cell numbers.
Best,
Le
Keep in mind that in all cases (MTT, LDH, cell titer glo, CCK-8) you are measuring metabolic activity as a proxy for number of cells. If you treat the cells with a compound which stops cell division, for example, but the cells continue to grow (get larger), not unlike senescence) you will miss the event. You need to either count traditionally as you originally described or use an assay which measures DNA content (which doubles with each cell division).
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