I have pated 1 million cells on a coverslip in a 6 well dish to perform immunocytochemistry. Now I have to plate the same in a normal 6 well dish to study gene expression. Please suggest to me how many cells I have to plate in a 6-well dish for expression studies. Please anybody can explain the calculation procedure briefly?
The calculation procedure will involve basic math. Determine the area of the coverslip and compare that to the area of the dish. Use that to calculate the number of cells needed to be plated on the larger surface.
For example, if you determine that the area of the well is twice the area of the coverslip, then you will need to double the number of cells to get the same concentration on the larger surface.
For culturing purposes we use 20k cells /cm square. Since you're seeding cells into a 6-well plate you should make your calculations according to the area of the well, not the coverslip. Because your cells will attach both onto the surface and the coverslip, so the total surface area will be the well's surface area. This is same for the gene expression studies as well. I believe using 1 million cells in 6-well plate may be quite too much for ICC. If you seed too much cells, cells may overlap and this may cause you to obtain false positive results. Make sure you perform ICC before cells get %100 confluent. And if you're not using poly lysin on your coverslips ı recommend you to coat your coverslips with poly-L-lysin or poly-D-lysin before seeding. It will help you to keep your cells attached to the coverslip during ICC manuplations.
Agree with Tayfun, suspension cells confluency should be visualized under microscope
How many coverslips in each well? Some times I put 1, sometimes 2 and rarely 3 coverslips. So, it's dependent to you to what do you want with the coverslips and remained cells in the wells. However, one full 10 cm2 plate is enough to seed up to four 6-well plates.
Manjari S K V I don't get your concern about the cell seeding number for both ICC & gene expression here by the way. Does it need to be the same for the number of cells that you have for ICC and QPCR? I am doing ICC and gene expression as well hence I hope we can have more discussion.
Regarding the calculation, not sure if I were right, but surely not all the 1 million cells are going to attach onto the coverslip. Let's say if only 50k cells are attached onto coverslip, then you should just plate 50k cells in the 6-well for gene expression. This is contrast to what Ron Hrstka has suggested. Here is another question, how to know how much of cells are really attached onto the coverslip? Then this question goes back to what is your concern of having same cell seeding number for both ICC & gene expression. Perhaps this should not be something to be concerned about, unless the results from ICC and gene expression are correlated to each other, which I personally do not think so.
Tayfun Dikmen I understood the density of cells to be plated. But in my case, I am going to plate the cells for the sake of differentiation. Please help me n this respect.
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