This article describes a method for assessing phagocytosis of antibody-opsonized sheep red blood cells (SRBCs) by macrophages. The assay utilizes complement proteins to facilitate the binding of SRBCs to macrophages, allowing for quantification of phagocytosis through hemoglobin detection.
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 C5-deficient human serum for 30 minutes at 37 degrees Celsius to fix the C3b and C3b inhibitor complement fragments on the IgM-coated SRBCs.
Next, aspirate the media and add 100 microliters of 1 times 10 to the seven opsonized SRBCs per milliliter of medium to each well of a 96-well plate containing 1 times 10 to the fourth overnight-cultured murine macrophages per well.
Incubate the SRBC mouse macrophage coculture for 1 hour at 37 degrees Celsius and then remove the unbound SRBCs by washing with 100 microliters of ammonium chloride-potassium lysis buffer for 1 minute.
After the unbound SRBCs have been removed, rinse with 100 microliters of media. Lyse the remaining cells with 0.1% sodium dodecyl sulfate and treat the lysates with 50 microliters of 2,7-diaminofluorene supplemented with 3% hydrogen peroxide and 6 molar urea.
Then measure the absorbance of the hemoglobin-catalyzed fluorene blue formation on a spectrophotometer at 620 nanometers.
Use a standard curve at 620 nanometer absorbance values with known number of SRBCs to determine the number of opsonized SRBCs that are phagocytized.
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