starting HaCaT cell line. basic questions - (Sep/27/2012 )

Hello everyone,
I am planning to start working with a Hacat cell line, need some advise regarding maintaining protocol. have scanned the literature regarding the media and all the suplements that peopel have been using. now there are some questions that i'm a bit confused about.

What i am planning to use:
DMEM medium: High Glucose, HEPES, no Phenol RedHigh (Invitrogen) + low conc, Ca2+ (0.06mMol)
FBS (not sure which would work best)
Gentamicin/amphotericin

Trypsin/EDTA (leaning towards 0.25%) though different protocols mention different %

now the question is regarding NON Essential Amino Acids . Found that some have been using some not. So here is the question, should that be in the medium or not ? your opinion or experinece.

did i forget anything?

Thank you in advance
R.

They are usually cultured with the non-essential amino acids (NEAA) - however, if you are getting them from a source (another lab) that isn't using the NEAAs, then you should ask them why they aren't and use the same medium composition that they are using.

You can wean the cells back onto the recommended medium + additives over several passages, doing this abruptly leads to cell death and alters the cell population drastically.

thanks for the reply,
i'm actually planning to buy them from a company, so i guess i should follow their suggested protocol?

R.

Yes you should - you should use the medium that they are using and all the supplements that they recommend for the cell line.

Thank You
R.

Hi Reiven, I have been culturing the Hacat cells using DMEM and 10% FBS together with glutamine since July. Nothing really special to culture this cell line and they grow nicely. Now they are kept in LN2. I've got mine from Addexbio. Good quality and high density. Where are you getting yours.

Hi h9494036
we are thinking of buying the cells from CLS (germany) for number of reasons.
and thank you for the advide on medium.

R.