JoVE Encyclopedia of Experiments
Microbiology
0 views • 3:22 min • January 30th, 2026
Take IgM-opsonized sheep red blood cells (SRBCs) with IgM antibodies bound to the cell-surface antigens.
Add serum containing complement proteins except C5.
The C1 complex binds to the antibodies and becomes active.
Activated C1 cleaves C4 and C2, forming C3 convertase, which then cleaves C3 and deposits C3b on the SRBCs.
Without C5, C3b degrades into C3bi.
Add the SRBCs to a macrophage culture.
C3bi binds to the macrophage receptor, triggering SRBC phagocytosis.
Add a hypotonic solution to lyse and remove non-internalized SRBCs.
Add lysis buffer to lyse the cells and release SRBC hemoglobin.
Introduce a detection reagent.
In the presence of urea, hemoglobin catalyzes the oxidation of diaminofluorene by hydrogen peroxide, producing a colored product.
Measure the absorbance of the colored product, which is proportional to the SRBCs phagocytosed.
This assay models phagocytosis of antibody-tagged bacteria using opsonized SRBCs to mimic bacterial targets.
For an opsonization assay, incubate 2 times 10 to the eighth sheep red blood cells or SRBCs with 50 microliters of rabbit anti-SRBC immunoglobulin M or IgM for 30 minutes at room temperature.
Then incubate the opsonized SRBCs with 50 microliters of
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