I am doing infection experiments with Mycobacterium smegmatis and when washing the macrophages at 4 hours post-infection they tend to detach from the plate.
Everytime J774.A1 cell line is having this problem, best way is to culture this cell line in the flask or switch to J774.A2 cell line.
Thanks. Do you know what is the difference between J774.A1 and A2 cell lines?
J774.A1 cells appeared to have less phagocytic ability and were not as efficient in migrating towards a positive control (c5a) when compared to J774.2 cells.
J774 cells are not a "real macrophages". As you know is a cell line cultured from long time ago and there are many sublines. Consequently, my recommendation is: (i) wash away those cells do not firmly attached; (ii) establish a "new line" and subcultured several times with only those adherent cells and (iii) repeat this procedure as many times as required in order to obtain an "adherent subline".
I hope this could be useful for your purposes.
¡Buena suerte!
In this case it is unlikely to be the bacterium causing a problem as Mycobacteria are so slow growing-so it is likely to be the cells. As Francisco correctly says J774 are not 'real macrophages' and with most transformed macrophage-lke cell lines long-term culture and the process of transformation leads to phenotypic changes.
The simplest suggestion is to go back to 'first principles' and re-start the cells from frozen stock-long term passage can be a problem. Grow on for a few cycles then try again. It is possible the Mycobacteria are changing the cell phenotype-if this is a consistent thing try another cell line (RAW) or primary macrophages derived from bone marrow. Good luck
Muchas Gracias to all of you, I will put in action all your suggestions.
What type of culture vessel are you using? Tissue culture-treated plasticware results in better attachment than non-treated plates.
I had been working with this cell line a while ago, and I have noticed the same thing. I used this line in Ag processing and presentation experiments, and when they detached they stopped presenting Ag. This was accompanied with the significant loss of MHC-II expression. So I would just throw out the old culture and start a new one. They seem to grow nicely for about 6 months, after which time they tended to detach.
I have use the J774.1 for phagocytosis of Raji for 4times, in the first phagocytosis, few phagocytosis were found under microscope. however, in the following several experiments, no phagocytosis events were observed, enen though we recoveried the frozen cell. Dose there any publications of comparation of cell line J774.1 and J774.2 ?
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