macrophage cell line - problem with the media conditions (May/11/2006 )

hi all ! iam posting this query for the second time with a hope to get some suggestions.
iam culturing J774 mouse macrophage cell line. i use DMEM (low glucose 1000mg/L) + 10% FBS supplemented with antibiotic and antimycotic solution.
i heard that using antimycotic and antibiotic solution will interfere with signal transduction studies. also can you tell me why use of high glucose (4500mg/ L media) is forbidden in the case of J 774. when i used high glucose, cells in the control wells ( with out infection with the pathogen under study) multiplied rapidly compared to the cocultured cells ( with infection with the pathogen under study ) which stopped multiplying after phagocytosis .
i would be thankful for sharing your experiences with this cell line
thanks in advance

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Dear Shiva,

1. Always use the 1000mg/L DMEM, I have been growing these cells for nearly 20 years and they grow like weeds. I have even gone down to 5% FCS to try and reduce the numbers of times/week I have to passage the cells.
2. We have also used these cells for studies investigating Salmonella infections. Again at MOI ranging from 1-100, the J774 will not proliferate.
3. Never use antimycotic/antibiotic in your cultures. If you have good technique then you should never get an infection with this particular suspension culture.

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I am using the J774 cell line for intracellular killing assays with S aureus. I find these cells are very fragile and do not grow continuasly for many passages. Why is it recommended to use low glucose for this cells? I have been using hi glucose DMEM or RPMI with 10% FBS . I also find problem in their revival rate when taken out from LIQ nitrogen CAn anyone help
lakshmi

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Dear Imundkur,

Your cells have a serious problem.........J774 cells are the toughest cells I have ever worked with and are almost impossible to kill. They grow like weeds in the garden and can be passaged over 200times......In fact my record was 216 passages over a 2 year period without contamination or changing the cells. The iNOS enzyme that I extracted from these cells was expressed at the same levels at passage1 to 216. Viability should be 99%.
The viability described above is J774 that were grown in SUSPENSION. If like most people you grow your cells as adherent cultures you have a problem. The cells will stick to the TC plastic like SUPERGLUE. The trypsinisation process can and will damage the cells. By growing them in suspension gets round this altogether.
If you are having problems reviving the cells from frozen then the freezing process may have gone wrong. Buy some more cells and start again.

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This may be a stupid question, but I hope Rhombus or someone else would answer it. How do you grow your cells as adherent, as opposed to in suspension? I always thought that the stickiness of the cells in a culture is a function of the surface properties of the cell itself, you know, like adherent- or non-adherent cell lines. I did not know that this behaviour could be modified in some manner. I would be obliged if someone could tell me how.

I use J774 cells in phagocytosis assays; I lay the cells down in the wells and overlay them with the bug. I would be definitely interested to know about this distinction in the culture method, because the cells in suspension may have different properties than adherent cells.

Cheers!

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Rhombus actually answered the same question for me on a different thread. Have a look: link

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