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

1. 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 before proceeding to the next step. Normally, 1-2 10-mL vacutainer tubes of whole blood are sufficient for the assay.
2. Dilute the whole blood 1:1 v....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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