Can we evaluate the phagocytosis of peritoneal macrophages in relation to Candida albicans, Gram+ and/or Gram- bacteria? Is it better to evaluate in vivo or in vitro assays? And by cytometry? If you have any indication of protocols that I can analyze, I would also appreciate it.
Kehinde Oluwasegun Aina
I will generally recommend Fluorescent Labeling and Flow Cytometry for (Quantitative) analysis
- Firstly Label bacteria or fungi with a fluorophore (e.g., FITC, pHrodo) and incubate with macrophages.
- How to distinguish internalized vs. adherent: Use quenching dyes (e.g., trypan blue or anti-FITC antibody) to remove surface-bound fluorescence. Or use pH-sensitive dyes like pHrodo, which fluoresce only in acidic phagosomes.
- Readouts can be : % phagocytic cells, mean fluorescence intensity (MFI), phagocytic index.
second suggestion will be to perform the Colony-Forming Unit (CFU) Assay (Functional Digestion)
- Procedure: After incubation, lyse macrophages and plate the lysate on agar.
- : Measure viable internalized microbes.
- Readouts: Lower CFU suggests better microbial killing (not just uptake).
- Useful for: Differentiating phagocytosis vs. bactericidal capacity.
Lastly, you can also check the assays for macrophages indicated in this paper , Article Generation and Functional Characterization of Monocytes and ...
All the best
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Sumara Freitas
Thanks for the information and the article! It will help me a lot! Kehinde Oluwasegun Aina
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