I am trying to find out how much cells are viable after my incubation. I am having issues with working out the standard curve.
Seed the cells in 96 well plate 'v' bottom and transfer to a flat bottom 96 well plate for the absorbance reading after incubation
How much cells should be ideal for this assay ? Currently using 100000 cells
Cell type : Jurkats
Seeding in the morning and reading done at the end of the day
It depends on your cell viabilty and metabolism. I detected FACS sorted mouse primary CD4 T cell. 50000 cells is enough for my CCK-8 signal (compare to negative control----medium+CCK8). In addition, time of CCK8 incubation and cell culture should be optimized.
Hi Xingchao Pao, can I know how do you prepare your standard curve ? I would appreciate if you could include some details.
So you use CCK-8 to detect what? standard curve for cell number counting? or cell proliferation or cell viability?(drug cytotoxity?). for cell proliferation or cell viablity, I didn't prepare standard curve. for this part, you should confirm the cell seeding number at the begining, time of cell culture, and time of cck-8 incubation in a preliminary experiments. OD450 larger than 0.5 will be more suitble. Standard curve between cell number and OD450 I checked as follows: for suspension cells, 5000, 10000, 20000, 50000, 100000, 200000 were respectively seeded into 96-well plates. 6h after seeding, add CCK-8 for another 2h. and read CD450. Similarly, here "6h","2h", you should also optimized in your system. It depends on your cell type and the purpose of experiment. Hope these will help for you!
Hi Xingchao Pan, the standard curve is for cell number counting
btw what type of 96 well plate did you seed in ? flat or round bottom ?
Standard curve for cell number counting is described as above. I use Corning CLS3599 96 well plate which is flat bottom.
hello ,The appropriate number of cells depends on the type of cells and the type of your experiment. The amount of coloration will differ depending on the cell type, even if the cell number per well and incubation times are the same. When
using a 96 well microplate, please check the absorbance level of 1,000-25,000 cells/well. If the experiment is for toxicity tests, 5,000-10,000 cells/well may be appropriate. If the number of cells are expected to increase during the assay,
prepare a plate with 1,000-5,000 cells per well.
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