I have to perform permeability assay by virus infection on endothelial cells. For this I need to use MOI 0.1,1, 10. For this purpose, I need to seed cells on the insert and count them before infection for calculating the MOI. For the usual experiments, we count cells and seed and then recount the cells just before infection from an extra well in the plate. So, how do I count the cells and estimate the MOI for infection once the cells are seeded?
Thank you for the reply. My doubt is that when wen keep it for attaching, firstly, all the cells will not get attached and secondly, they will be diving in the next few hours. So, our ultimate cell number estimated for a MOI calculation changes. How to deal with this?
I think using a control well to recount the cells is a good idea to estimate the number of cells that divided , attached or not.
yes it is a good way to know the approx count of cells but when we seed on an insert. it is a permeable membrane support on which fibronectin coating is done before seeding the cells. So, how do we count on it??
I think the two answers above have covered your only option. 1) Seed as many cells as you need and infect them about 6hr later once they have attached and before they begin dividing. 2) Seed a extra insert and then count the cells on that to use as you cell number. You are only ever going to be able to approximate in these experiments. This is why you repeat experiments, to normalize the error that you get from counting. A third option would be to use something like a high content analysis machine. You could stain for cells with a DNA dye such as Hoechst 33342 and then use the analysis software to count the number of live cells. This is a complicated and long way to count cells. There is also the chance for some toxicity due to the Hoechst 33342 dye. I have always seeded and extra well or slide and counted this to get my cell number and this i the accepted method in virology.
- Badges
- Science method