I am going to incubate suspension cell lines with cell-penetrating peptides carrying a pro-apoptotic agent. and then I have to wash them 2 or 3 times to remove those peptides that do not entered the cells. then I should stain the cells using propidium iodide for death assay by flow cytometry.
according to the point that I have to wash these cells again after adding PI, I will lose a lot of cells by continuous washing.
I need at least 100,000 cells remaining in each well for flow cytometric analysis...
so how many cells do you suggest me to seed?
I am limited to use the peptide. and I have to use 96 well plates with just 100 uL total valume.
The number of cells to be seeded in any plate or well depends on three things
1. Rate of growth of the cells
2. Incubation duration
3. Final cell count needed.
I normally suggest not to go for more than 1,00,000 cells per well in a 96 well plate. However, if the incubation time is too long or the cells grow faster, you need to go down further in seeding the cells. You can increase the culture volume to 200 microliters if you want to increase the cell number.
Since your final readout is by flow cytometry, you do not need to acquire more than 10,000 events per sample. So 20,000 cells are enough in your sample.
If you are worried about losing cells during wash steps, use V-bottom plates, which form tight pellet for your washing steps. I assume there is no problem since you are working with suspension cells.
I hope this information is helpful to you. Goodluck
10,000 - 50,000 cells in 200 micro litre to each well plate will be sufficient enough to get the data.
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