Home › Biochemistry › Use of a Monocyte Monolayer Assay to Evaluate Fcγ Receptor-mediated Phagocytosis
Biochemistry JoVE (Open Access) Citable · DOI

Use of a Monocyte Monolayer Assay to Evaluate Fcγ Receptor-mediated Phagocytosis

DOI: 10.3791/55039-v
What you'll learn
  • ✓ Isolate primary monocytes from peripheral blood using density gradient centrifugation
  • ✓ Opsonize red blood cells and measure Fcγ receptor-mediated phagocytosis in vitro
  • ✓ Quantify phagocytosis efficiency using monocyte monolayer assay methodology
Protocol

The monocyte monolayer assay (MMA) is an in vitro assay that utilizes isolated primary monocytes obtained from mammalian peripheral whole blood to evaluate Fcγ receptor (FcγR)-mediated phagocytosis.

Difficulty
intermediate
Total time
~4–6 hours per blood donor sample (PBMC isolation ~2 hrs; assay setup & incubation ~2–3 hrs; analysis ~1 hr)
Model organism
Primary human monocytes from peripheral blood
Biosafety
BSL-2

Steps

1
Isolate peripheral blood mononuclear cells from whole blood

Obtain mammalian whole blood and perform density gradient centrifugation to isolate PBMCs containing monocytes. This preparatory step yields the primary cell population required for the assay.

▶ 00:58
2
Opsonize red blood cells and evaluate phagocytosis

Treat R2R2 red blood cells with antibodies to opsonize them, incubate with monocyte monolayers, and quantify Fcγ receptor-mediated phagocytosis using microscopy or flow cytometry. This step directly measures receptor engagement and cellular uptake.

▶ 02:22
3
Analyze and quantify monocyte phagocytosis results

Process representative data from monocyte phagocytosis assays to calculate phagocytosis indices and compare experimental conditions. Results interpretation demonstrates how to assess FcγR function from microscopic or cytometric readouts.

▶ 04:16
💬 Comments coming soon