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NIH3T3 cells are suddenly dying a few days after thawing and expanding - (Dec/23/2011 )

Hello. I am new here, so please bear with me. I have recently been trying to culture transfected frozen NIH3T3 cells. There are two lines of the transfected cells: with an empty MSCV vector and one with the gene of interest. Both have hygromycin resistance. After thawing the cells, i have added 5ml of media, spun them down and resuspended in 2ml of media and put into a well of a 12-well plate. I have expanded the line with the empty vector into a 10cm dish after a few days of growth. They have been growing slowly, but steadily. I changed the media yesterday and today almost all have died after steady growth of a few days. The cell line with the gene of interest vector is still in the 12-well dish and seems to also be dying. I don't understand why this is happening. These are the second vials I have thawed for each line after they died the first time and it was the gene of interest line dying in the 10cm dish after a few days last time.

My media is DMEM with 10% FBS, P/S, L-glut and HEPES. I have used it in another NIH3T3 line and with HeLa cells and they seem to be doing fine.

Any help would be greatly appreciated. Also, if needed, I can provide more details.

Thank you,


I presume these are stable transfections, not transient?

When do you add in the hygromycin following thawing? It is usually best if you leave it out for 24-48 hours, so as to give the cells time to recover and start expressing the resistance gene again. You should also check the level of hygromycin used. For maintaining the cells you should only use 1/10th -1/2 of the level used for selection, as it should only be required to keep the plasmid integrated, it's also cheaper.

It may pay to check which medium the cells were grown in before freezing, you may find that they were in some other medium, in which case you should wean them onto your DMEM.