6 replies to this topic

[Maria]

Hi there,

If someone can help me, I would appreciate it!!

I am going to be growing up Hep G2 cells and I have had no experience with them at all. So my questions are:

How many times can you passage them?
How do you freeze them down?
Do you need any additional supplements to differentiate them ?
How do you check their phenotype?

  too many questions huh!

Thanks

Maria

[Maria]

  

Any ideas would be great :-)

[fred_33]

hi
well for freezing cells, you can see the topic freezing and thawing of cells. As i know, there is no specific protocol for hep G2 cells.

For the limit of passages, my colleague who works with hep G2 cell line do not informed me about a limit. I suppose therefore it's quite an immortal cell line.


for specificities : i found at ATCC

The cells express 3-hydroxy-3-methylglutaryl-CoA reductase and hepatic triglyceride lipase activities.
The cells demonstrate decreased expression of apoA-I mRNA and increased expression of catalase mRNA in response to gramoxone (oxidative stress).
There is no evidence of a Hepatitis B virus genome in this cell line.
extracted from this webpage

hope that helps you
fred

Edited by fred_33, 23 March 2005 - 08:41 AM.

[BryanW]

Be very careful that some differentiation methods also potently induce HepG2 apoptosis....2% DMSO or retinoic acid should do the job.

2 Markers for HepG2 differentiation: (1) Alkaline phosphatase can only be found in well differentiated human hepatocytes (2) albumin is a protein secreted by mature hepatocytes.

[Maria]

Thanks for the advice :-)

[Chantal]

Hi Maria:

I have been growing HepG2 cells for 4 hears.
You can passage them 20-22 times. I usually thow new cells when the old are at passage 16, therefore I do not use HepG2 after passage 20.

Freezing:

Tripsonize cells at 80-90% confluency.
Add 12ml of RPMI-1640 (Invitrogen) + 1% Glutamine to resuspend the cells.
Spin the cell suspension at 200g for 3 min.
remove supernatant.
add 7 ml of freezing media (composition of freezing media: 10% DMSO, 20% FBS, 70% RPMI-1640).
breack cells clumping with syringe once.
Aliquote in cryovials (1ml in each vial).
put vials inside NALGENE Cryo 1C Freezing Container and put at -78C for 24 hrs (cells must freeze 1C/min).
Remove from Freezing container and store vials at -78C or in liquid nitrogen.

[Maria]

Thanks alot Chantal  

Chantal, on Mar 25 2005, 02:09 AM, said:

Hi Maria:

I have been growing HepG2 cells for 4 hears.
You can passage them 20-22 times. I usually thow new cells when the old are at passage 16, therefore I do not use HepG2 after passage 20.

Freezing:

Tripsonize cells at 80-90% confluency.
Add 12ml of RPMI-1640 (Invitrogen) + 1% Glutamine to resuspend the cells.
Spin the cell suspension at 200g for 3 min.
remove supernatant.
add 7 ml of freezing media (composition of freezing media: 10% DMSO, 20% FBS, 70% RPMI-1640).
breack cells clumping with syringe once.
Aliquote in cryovials (1ml in each vial).
put vials inside NALGENE Cryo 1C Freezing Container and put at -78C for 24 hrs (cells must freeze 1C/min).
Remove from Freezing container and store vials at -78C or in liquid nitrogen.

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