J774 Macrophage - (Feb/28/2007 )

Hi all,

I am trying to use J774 macrophage for phagocytosis model. According to the literature, the cell is needed to induce by PMA for 72 hours to change from monocyte to macrophage. I am now working it by inducing the cell in flask for with PMA 48 hours first (add fresh medium after 24 hours), then subculture the cell by scraping the cell to 24-well plate with desired conc. and induce for 24 hours more. After wash with fresh medium, the cell will be used for phagocytosis.

However, I found that not all the cell are induced to become macrophage and some of the uninduce monocytes will still keeping growing.

Does anyone have better protocol on induction of J774 with PMA.

Thanks

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I am also trying to investigate phagocystosis with J774 macrophages. Are you growing the cells in suspension or letting them adhere to a surface when growing? Also, where did you find the information about converting them to macrophages? Do they not already come as macrophages?

Thanks

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Laxman is totally correct, J774 are macrophages. You do tend to get two different morphologies with these particular cells. If you seed them onto plastic you will see:
i) Round golden cell morphology.
ii) Fibroblastic looking cells.

We use these cells to produce iNOS through LPS treatment (10ng-10ug range). When you visualise the cells not all the cells induce. But this does not correlate with morphology.

You should be growing these cells in SUSPENSION and NOT scraping them to subculture...this causes too much damage to the cells. I have sent Laxman a private message with all the information he required about growing the cells in suspension. As you well know J774 stick to the TC plastic like SUPERGLUE...hence the scraping.

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Hi
I'm intending to use J774 cells to infect them with apicomplexan parasites and compare cultures in suspension vs. adherent, would anyone mind giving me some input about culturing J774 in suspension? may I culture them in a T75 flask using agitator or is it neccesary to use stirrer flasks? Is somethere a protocol I could try on? Thanks in advance

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I' am also culturing J774 macrophages. I use high glucose DMEM with 10% FBS in spinner flasks. Right now, I split by centrifuging the cells, pipetting off the old medium and resuspending the cells in fresh. Yesterday the pellet was a weird color. What color should it be? Also, is there a better protocol I can use for passaging the cells? I'm new at this and my PI's a physicist, so I really need help.

Thanks

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No need to use the high glucose DMEM, use the 1000mg/litre DMEM. Also the cells grow very fast so you can reduce the FCS concentration down to 7.5/5%.
I use 500ml stirrer bottles. To subculture take off 498mls of cell suspension and then refeed with fresh media. In 3 days your cell density will be back upto 750,000 cells/ml. This is the optimum cell density. If you go above this then the cells do suffer i.e. your viability will drop from 99%.

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Thanks!

If I want a set of adherent J774, can I use a T75 with TC treatment?

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Hello :
I am trying to use J774 to infect with Leishmania. My problem is that my cell growth very well in suspension in RPMI. However, when I try to plate the cell into 24-well plate, the cells stay in suspension! They adhere to the surface for 3 h but after that all cells stay in the middle of the well and not attached!. Please, I will appreciate any advice.
Thanks

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Stimulatino with PMA/etc can make them more "macrophage-like" and stick much better to plastic.
Personally I don't trust the un-stimulated results - the cells behave very different in regards to signalling pathways/cytokine production etc
Just remember to factor in the difference. Either way is fine as long as you control for it.
Do you have to do the phagocytosis/infection on adherent cells ? can it not be done in suspension? (certainly for FACS, if for microscopy what about cytospinning the cells to glass after? - not sure if they remain intact enough.)

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