The idea is show drug of interest is not affecting functional ability of these
I'm thinking along the lines of:
1. Stimulate primary macrophages (or just Raw 264.7 cells) with LPS and treat with drug of interest. Label the macrophages and run on flowcytometry.
It doesn't sound right to just do this or may be I'll have to test for engulfment of FITC zymosan beads etc as the idea is to test the ability. Any thoughts?
2. Activate B cells with LPS/IL4 and treat with drug of interest, Measure IgG1? I'm not sure if this is called quicker as minimum activation period is 12-24 hrs. Any other way? IgM would be quicker ?
Hi Arathi,
Flow staining of B cells cultured with IL-4 and LPS for 48 hrs is quickest. Look for IgG + ve B cells. Along with IgG, CD27 is also upregulated on activated B cells. You may also save sups from this cultures to perform IgG, IgM ELISA. More IgG suggest better activation. I don't work with Macrophages so no comment for these cells. However, increase in expression on MHC-II (mice) or HLA-classII (humans) should indicate activation of macrophages. Hope this helps.
For macrophages you can try a shor-term stimulation with LPS or PMA.
There are various ways to test the functionality of these cells.
One way would be to test their cytokine response. If you have a pure macrophage culture, i think a multiplex assay from the supernatant to check the levels of various cytokines would be the easiest.
However, if you have a mixed culture such as PBMC, a supernatant assay won't tell you much about the cytokine response of either population. In this case, a cytokine intracellular staining assay coupled with surface staining could show you exactly if and how many of your macrophages are secreting the cytokine(s) of interest. Although it can be pulled off in less than one day of work, this is a more elaborate assay.
The cytokine response however is not the main role of macrophages. I think that the best way to check if your macrophages are still functional is to test their phagocytosis capacity (of course). There are various protocols in the literature, but i will leave one link here.
https://www.researchgate.net/publication/292565393_Culture_and_Differentiation_of_Monocyte_Derived_Macrophages_Using_Human_Serum_An_Optimized_Method
Look at the methods section, 3.4 and 3.5. Such assays are way cheaper than flow cytometry which requires expensive antibodies and you can also see the cells' morphology and take photos.
Last, i don't know what drug you are going to use, but i just wanted to add for the record that, depending on the mechanism of action of that drug, you may need to incubate the cells with the drug for a while BEFORE you do the functional assay. Simultaneous exposure to the drug and stimulation may not reflect the true inhibitory potential of that drug on macrophages...but leave them 24h with it and you may find them dead or with highly alteres functionality.
For macrophages you may perform phagocytosis assay using fluorescent labelled E. coli or S. aurious, which takes around 60-80 minutes and check on flowcytometry.
Or you can check oxidative burst by Flowcytometry, it also takes around 45-60 minutes.
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