Source: Tong, T. N., et al. Use of a Monocyte Monolayer Assay to Evaluate Fcγ Receptor-mediated Phagocytosis. J. Vis. Exp. (2017)
This video showcases a monocyte monolayer assay utilizing primary monocytes from peripheral blood to assess Fcγ receptor-mediated phagocytosis with poly D antibody-opsonized R2R2 red blood cells.
All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.
1. Peripheral Blood Mononuclear Cell Isolation
2. Pre-treatment of Adhered Monocytes
NOTE: This step is only necessary if looking for ways to inhibit or enhance phagocytosis.
3. Opsonization of R2R2 Red Blood Cells
NOTE: Opsonized R2R2 RBCs are used as a positive control for FcγR-mediated phagocytosis. Naïve R2R2 should be stored in Alsever's solution (to prolong shelf life) at 4 °C for up to 1 month. Alsever's solution is made in-house and composed of 0.8% w/v trisodium citrate (dihydrate), 1.9% w/v dextrose, 0.42% w/v sodium chloride, and 0.05% w/v citric acid (monohydrate). If R2R2 cells are not available, other Rh-phenotyped cells, such as R1R2, R1R1, R1r, or R2r, can be used.
4. Fc Receptor-mediated Phagocytosis
5. Quantification of Phagocytosis
The authors have nothing to disclose.
RPMI 1640 | RPMI 1640 | R8758 | |
Phosphate-buffered saline | Sigma | D8537 | |
8-chamber slides | Lab-Tek-ll | 154534 | |
R2R2 (cDE/cDE) red blood cells | Commercially available (e.g., http:// www.bio-rad.com/en-ca/product/ reagent-red-blood-cells) | ||
Polyclonal anti-D from human serum | Gamma Biologics | DIN 02247724 | Can be substituted with commercially available monoclonal anti-D or with Rh immune globulin |
100% methanol | Caledon | 6700-1-42 | |
Cover slips | VWR | 48366 067 |