Preparing a cell culture stock to plate out at 7x10^5 per well? - (Nov/07/2013 )
I have a stock media of cells (THP-1), as of yet unknown cells per ml.
I want to make a stock solution which contains 7x10^5 cells per well, each well will have 2ml of media added. So 3.5x10^5 cells per ml.
I have a total of 72 wells to prepare, so 148ml required, but I will make up more in-case of any pipetting errors. Say 150ml or 160ml.
So to start with I will extract 1ml from the stock media with cells and from that take 10ul and transfer to a haemocytometer to count the number of cells per 10ul then convert to cells/ml.
Once I know how many cells per ml I have, I will then need to calculate the volume of media with cells I need to spin down in the centrifuge, which will then be re-suspended into stock media with no cells so that I have the correct concentration of cells to pipette out into the wells.
If possibly could you give an example of how I would proceed through each step.
You have just outlined the steps you need to do, so I don't see where your problem is. If you need counting calculation examples, look through the many (many) threads on here about counting/calculations.
Incidentally - you don't need to spin down the aliquot of cells you take, just add them to a volume of medium that will make up to your final volume. For example, if you need 6 ml of your cell suspension to get the required cell number and you need to make the final volume 150 ml to get your desired cells/ml and enough to plate out 1 ml/well, just add 144 ml of medium to the 6....