Newbie help: Seeding cells to 6-well plates
Posted 28 November 2011 - 02:49 PM
I'm a new volunteer at a lab and I lack experience, so I have this simple question. I have asked my mentor but she didn't explain it quite clearly, seeing if any help can be received from here.
I'm going to be doing cell transfections and needing to seed my 293T cells to 6-well plates. Am I on the right track?
Remove medium from current 10cm culture plate.
Wash with PBS, remove it, add in 1mL Trypsin to detach the cells.
Add 3mL DMEM to stop Trypsin action.
Take a small drop of cell/DMEM with a micropipette and put it into the hemocytometer to count the cells.
Count the 4 corners, count 2 of the edges of each square (ie: 85 total)
Calculate: 85 * 10000 * 4 = 3400000 cells in my 10cm plate (with 1mL trypsin and 3mL DMEM)
This is where I was a bit confused. We need about 80% confluency, and about 1.2 x 10^6 cells per well, with 3-5mL total volume. What my mentor told me was to just for example, put 2mL DMEM into each well and 1mL of cell solution. When it is confluent in a day, it'll be 1.2x10^6 cells in each well. That doesn't seem to make sense, as what would be the point of the hemocytometer then? My mentor's English isn't amazing, so maybe we just are misunderstanding each other.
My main issue is that for example, if I have 3400000 cells in my 10cm plate, how much of that do I put into each well?
If anyone can help me out, that would be greatly appreciated. I may (will) be coming back for more help later on.
Posted 29 November 2011 - 10:45 PM
Posted 02 December 2011 - 02:21 PM