An Assay for Studying Fcγ Receptor-Driven Phagocytosis in Monocyte Monolayers

Abstract

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.

Protocol

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines. 1. Peripheral Blood Mononuclear Cell Isolation Obtain human whole blood from a healthy donor or a patient via venipuncture using vacutainers containing acid-citrate-dextrose (ACD) anticoagulant (yellow-top tubes). NOTE: Whole blood can be stored in ACD at room temperature (18-22 °C) for up to 36 hours be…

Materials

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