Newbie help: Seeding cells to 6-well plates - (Nov/28/2011 )

Hi

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.

hola, if you have 3.4x10e6cells in 4ml only you have cells for seed 3 wells at 1.2x10e6 adding 1.3ml, but if you add 1ml as your mentor says you will seed 8.5x10e5 and you will need 2 days to get confluence, and if you seed 6 wells you need one day more. Your mentor says this because he has experience and knows that in all the cases confluence is a time question. Buena suerte

Use of C=n/V and CiVi=CfVf (where C= concentration, V=volume, n=number of objects and i and f are initial and final respectively) will help you a lot in learing how to do dilutions correctly.