Macrophage Based Assays
Macrophages are effector cells of the innate immune system that phagocytose bacteria and secrete both pro-inflammatory and antimicrobial mediators. In addition, macrophages play an important role in eliminating diseased and damaged cells through their programmed cell death. They regulate lymphocyte activation and proliferation, and they are essential in the activation process of T- and B-lymphocytes by antigens and allogenic cells. Enhanced bactericidal activity of “activated macrophages” is based on immunologically linked mechanisms involving lymphocytes.
Difference in phagocytic potential of M1 and M2 MΦ
Primary monocytes (CD14+) were isolated from PBMCs of healthy donors and were treated with M-CSF for 6 days. On day 6, the naïve macrophages were exposed to GM-CSF + IFNγ and M-CSF to generate M1 and M2 macrophages (MΦ) respectively. The cells were then cultured with fluorescent zymosan particles for 3 h (green-phagocytic marker). After 3 hours, the cells were washed and thenstained with a fluorescent membrane marker (red) and DAPI (blue-nucleus).
The images above showed M2 macrophages engulfed higher numbers of zymosan particles when compared to M1 macrophages (Objective= 20X).
Difference in phagocytic potential of M1 and M2 MΦ
The fluorescent images from the phagocytosis assays were analysed using ImageJ. Each dot on the scatter plot represents 1 image field at 20X objective.
[A] Percentage of Zymosan positive cells = (Cells with zymosan particles/ Total number of cells per field)*100; [B] Phagocytic Index = (Average number of particles per positive cell)/ Percentage of Zymosan positive cells
M2 Macrophages (n=9) showed higher phagocytic index when compared to M1 macrophages (n=5) [±SEM].
Phagocytosis assay using PMA-differentiated THP-1 cells
THP-1 cells (immortalised human monocyte model) were treated with PMA to induce macrophage like characteristics. Phagocytosis was subsequently assessed by incubating cells with fluorescent zymosan particles (green). The images were captured at 20x objective and analysed for phagocytic index using Image J (n=5±SEM).
Differentiation of primary monocytes to M0, M1 and M2 MΦ
CD14+ monocytes were isolated from PBMCs for macrophage (MΦ) differentiation. Monocyte differentiation to M0 MΦ (Panel A) was induced and inhibited following treatment with M-CSF and SB203580, respectively. Polarisation of M0 to M1 MΦ (Panel B) was induced and inhibited following treatment with LPS and Dexamethasone, respectively. Polarisation of M0 to M2 MΦ (Panel C) was induced and inhibited following treatment with IL-4/IL-10 cocktail and Tofacitinib, respectively. Characterisation of M0, M1 and M2 MΦ in the various conditions was analysed through expression levels of cell surface CD86, CD80 and CD206 markers using flow cytometry.
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