I would like do the experiment with Bone Marrow-Derived Macrophages (BMDM). So I need to do Phagocytosis assay. Could you share the phagocytosis assay protocol in this regard.
Thanks for your response. Actually I wanna measure macrophages with Phagocytosis assay. There are some way to measure, ex, FACS or IF, which one more accurate and easy? Plz let me know@ Shen-An Hwang
The easy to use is flow, it's the fastest and most quantitative. But the flow does not give you how many particles were phagocytosed per cell, the most it can do is give you intensity. So... depend on what you want your analysis to be. And one thing to keep in mind... keep your time point short... definately less than 4 to 6 hours. Any longer and the cell start to digest the stuff they phagocytosed.
I use the generic term uptake because unless you actually target the markers and look for these markers in the organelle for phagocytosis, it's difficult to know what mechanism is used. The easiest way to get true phagocytosis is to use a pathogen that is well know and well characterized to be phagocytosed. In my case, I used mycobacteria, such as BCG. And I just fluorescent labeled the bacteria.
The easiest material to look at for uptake is fluroescent beads... but make sure your beads are at least 5um. The smaller ones tend to under go endocytosis.
there are numerous easy protocols to study phagocytosis of macrophages. You can simply incubate macrophages with FITC labelled dextran or Zymozan for 30 min at 37°C. You can use macrophages + Dextran-FITC at 4°C as control. You can determine the phagocytic activity by flow cytometry or confocal microscopy.