Serum withdrawal in HepG2 cells

[evilhamster]

hi i was wondering if culturing HepG2 cells without serum for 24 hours will drastically affect the cell in any way. I was performing glucose depletion experiments in the media and thought about how FBS causes spectral interferences in assays so i tried culturing my cells without serum for 24 hours. Will the cell be affected in any way? On the note of the FBS having insulin and other growth factors as some literature may suggest, i know that the FBS is heat inactivated so the proteins may either be denatured or whatnot? help please.

P.S. the hepg2 cells were grown to 100% confluence before serum withdrawal.


thanks,
evilhamster

[admin]

Hey,
we have serum starved HepG2 cells without FBS for several days although this stresses the cells. Some people have published studies growing without FBS for a long time (HEPG2 cells more than a month with other factors).

Keeping your cells at 100% is maybe not the best idea, are you trying to get them confluent for a reason?

[evilhamster]

i was under the impression that 100% confluent cells grow less, and that around 95-100% confluent cells are ideal for glucose depletion measurement in the media. This is because my hepg2 usually stay at around 50-60% confluent after 1:2 splitting for about a week. I've tried using insulin transferrin selenium supplements and this seems to reduce the doubling time by 1 or 2 days. Unfortunately ive not heard of ITS being used for hepg2.

Can i start measuring the glucose depletion in the cell media (and for future reference flow cytometry) when they are in about 70% confluence? i feel that if i do this the cells may consume glucose due to the log phase which is what i am trying to avoid. thats why i culture my cells to ~100% confluence. I know that the cells may mutate or something, but how extensive can the mutation be if it is only left at 100% for 24hours?


This thesis is being such a pest because my hepg2 grow so slow.

[evilhamster]

on another note, i have been wondering if i REALLY need to deproteinize the cell media i collect for analysis using the Trinder method (glucose oxidase). Most kits use sera and plasma samples without deproteinization, while other sources use trichloroacetic acid (8-10%) to precpitate proteins. I was wondering if i still need to deproteinize cell culture media since the protein content is considerably lower than that of sera or plasma.

[wr_1a]

thanks