I see the newly thawed J774A.1 cells form clumps of about 10-20 cells. On sub-culturing the size of the clump reduced to about 5-10. Is that normal? I am using DMEM media supplemented with 10%FBS and 1% P/S. Cells are grown on T75 flasks.
Ramya,
Such clumping may result from cell activation as suggested above. In the situation you describe, this may have occurred during freezing or thawing. Alternatively, clumping may result from freezing over-confluent cells. When J774 become completely confluent, they will form clumps during transfer. Because they grow very fast, this will occur if the transfer is just one day late.
The solution may be fairly simple. When your cultures are about 80% confluent, do a transfer. While the cells are in suspension in a 15 ml tube, let the clumps settle to the bottom for 1-5 min without centrifugation. You will see them accumulate at the bottom of the tube. Then use the 'supernatant' to replate. The clumping should be greatly reduced in the replated cultures. This may need to be repeated in one or two subsequent transfers to completely remove the clumps. If the clumps recur, then there is some other problem that is causing ongoing activation.
Good luck!
The short answer is NO. These cells in culture attach to the culture plasticware surface and morphologically appear macrophage-like (see the link from ATCC below). Therefore, check medium for certain stress-inducing signaling in these cells.
Link: http://www.atcc.org/products/all/TIB-67.aspx#characteristics
As Dr. Choubey mentioned, no, J774A.1 do not grow into clumps in cell culture and they should be highly adherent. You should see J774A.1 in culture (that is, not activated) to appear as two distinct populations: one that is round and monocyte-like and the other that is stretched and more macrophage-like (maybe even fibroblast-like. Not spread, like a HeLa or epithelial cell, but elongated as if it were moving towards an object.) At higher densities, you will see much more of the round type, but at 40-80% confluence, I would say that the two populations should be highly distinct.
Attached is an image of a higher density population of J774A.1. These were healthy cells, but seeded more than I would have kept them at in culture because of the experimental setup. Even at this high density, you can make out some cells that are a bit more spread, but mostly these cells are rounder.
It is important that you passage these cells by scraping, not a dissociation reagent. What is your culture procedure?
In my experience J774A.1 do not tolerate some antibiotics (e.g. the mixture of penicillin-streptomycin-amphotericin B) and I had good results when I switched to antibiotics-free media. In fact, the routine maintenance of cells with antibiotics in media is generally against the principles of a good cell culture practice (for instance, it will not address the problem of mycoplasma infections, which are sometimes cryptic and sometimes cause sudden changes in cell cultures, such as reduced cell attachment, massive cell death, metabolic changes and other). As already indicated above, these cells needs to be dislodged by scraping and dissociation with trypsin needs to be avoided.
Ramya,
Such clumping may result from cell activation as suggested above. In the situation you describe, this may have occurred during freezing or thawing. Alternatively, clumping may result from freezing over-confluent cells. When J774 become completely confluent, they will form clumps during transfer. Because they grow very fast, this will occur if the transfer is just one day late.
The solution may be fairly simple. When your cultures are about 80% confluent, do a transfer. While the cells are in suspension in a 15 ml tube, let the clumps settle to the bottom for 1-5 min without centrifugation. You will see them accumulate at the bottom of the tube. Then use the 'supernatant' to replate. The clumping should be greatly reduced in the replated cultures. This may need to be repeated in one or two subsequent transfers to completely remove the clumps. If the clumps recur, then there is some other problem that is causing ongoing activation.
Good luck!
I am recently cultruing this cell line,J774A.1. ATCC and many reserchers advise not using trypsin to passage ,but I just do not why?Dose trypsin affect or activate J774A.1?Is there any article explaining why the trypsin is not recommended in J774A.1 cell line?
Thank you!
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