Help! How to prepare 1 x 10^6 cell - cell culture (Jan/05/2011 )

Hi Everyone,

I know my question is a very basic one. I'm still new in this tissue culture area. I just want to know how to prepare 1 x 10^6 cell from scratch, I mean I want to know the procedure step by step from the beginning. I'm using HepG2 and I 'm using petri dish culture plate to culture the cells.

I am really hoping there will be some reply.
Thank you in advance.

Hi!
I hope I could help you:
First rinse cells with 5ml PBS, after that add 1-2 ml of Trypsin-EDTA, put them in the incubator, after 2-3 min look them (microscope) and observe if they detach, if that doesn't happen, leave them 1-2 min plus. Add PBS (8-10 ml) and collect all (liquid+cells) into 15 ml falcon tubes. Centrifuge for 5 minutes at 1500 rpm and then discard supernatant and resuspend cells in 1 ml medium. Count the cells in a hemocytometer and add the medium necessary for having 1x10^6 cell/ml
Good luck

Note that RPM measurements are not useful at all for centrifugation unless you know the model for the centrifuge and the rotor used - for pelleting cells you should use 100-300 RCF (relative centrifugal force) which are convertable between centrifuges

Thanks IreneG and Bob1 !!

I would like to ask more It's about how to do the dilution. I've already culture the cells and harvest and also did the counting.
Now I have 4.46 x 10^6 cells/ml in 20ml media. How to prepare 1 x10^6 cells/ml for total 40ml?
Because I need to seed the cells in 3 plates of 6-well plate with 2ml for each well (that's mean I need 36ml for 18 wells but want to prepare extra 4ml for subculture).
And, is it possible to seed with 1 x 10^6 cells/ml in 6-well plate?
Or is the density too high?
I think I've already ask too many questions. Please forgive me

And last but not least, Thank you very much again !!!

For a concentration of 1x10^6 cells/ml in a volume of 40 ml you need a total amount of 40x10^6 cells. You cell suspension has a concentration of 4.46x10^6 cells/ml. So take 40/4.46 = 8.96 ml of your cell suspension and fill up to 40 ml.

Use C1 x V1 = C2 x V2
Where C= concentration and V= volume.

In your case C1 = 4.46x10^6, C2 = 1x10^6 and V2 = 40 (ml, keep units consistent!)

Therefore V2 = 1x10^6 x 40
-----------
4.46x10^6

V2= 8.96 ml of your cells + 31.04 ml of medium.

2x10^6 cells per well is a fair number, but it depends on the cell type, the purpose of the experiment and the duration of the experiment as to whether it is too dense a seeding. For cells like HEK293, A549, or HeLa grown for more than 48 hours this will be a problem as the cells will be very densly packed and they can behave differently when grown like that.

I have seeded as low as 100 cells in a well of a 6 well plate (for colony forming assays) and had them survive - it depends on the cell type and how you treat them.

Thank you very much EIHo and Bob1 !!!

Now I am fully understand how to do the dilution .
I will follow all the suggestions that been given by you guys
Thank you...thank you...Terima Kasih