Hi! I am testing different vaccine adjuvants and their interaction with RAW 264.7 mouse macrophage cells. I am specifically interested in phagocytosis of the adjuvants. After phagocytosis, cell will be analyzed using Imaging Flow Cytometry (IFC). There are publications out there where they look at phagocytosis of aluminum-based adjuants, but even when they mention whether the cells were co-cultured with the adjuvant adherent or non-adherent, they dont specifically explain why.
It would be great if I can get your opinion about this question. Thanks!
Hi, I've done these sorts of phagocytosis exp's with J774 cells using pHrodo Red E.coli bioparticles from Fisher. They are adherant cells, so I opted to first use a plate reader as these particles only fluoresce once inside the cell. This worked well, just add the beads, and take plate readings at various time points. I also used fluorescent microscopy with vesicle imaging and a DAPI stain to get fluorescence/cell and get nice pictures. Fresh J774's had significant phagocytosis, whereas LPS activated cells phagocytosed significantly less (as expected).
When I switched to looking at splenocytes (non-adherant) for phagocytosis I opted to use flow cytometry, this was great too, can classify the cell types and look at phagocytosis.
I didn't try using flow with the J774's because I was worried about behaviour changes of the cells being lifted off the plate after phagocytosis or if I lifted them before and kept them in EDTA or something to keep them from adhering I wasn't sure they'd behave the same with bead uptake. And it was just much easier to use the plate reader at say 15min, 30min and 100min post adding the beads, then fix the cells in the plate, DAPI stain them and eventually image with with microscopy.
Are you looking at the effects of alum activation on RAW cell cells and their phagocytosis potential, or are you specifically looking at alum in the phagosomes of RAW cells?
The cell line that you propose to use is adherent (see link below). However, loosely attached cells can be "selected" for certain experiments under certain conditions. However, these conditions need to optimized for reproducibility.
Link: https://www.atcc.org/Products/All/SC-6003.aspx#generalinformation
Thanks Divaker & Felicity!
Felicity, yes I am interested in looking at the amount and speed of phagocytosis of aluminum-based adjuvants by the RAW cells, not really at the activation of the cells at this point. I really like your idea of using a plate-based assay, where you just analyze the whole well. Have you published a paper with the methods?
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