Flow cytometry analysis of C3b surface binding and uptake in Trypanosoma brucei gambiense

Trypanosoma brucei gambiense take up human complement factor C3b by endocytosis via the specific surface receptor ISG65 (invariant surface glycoprotein of 65 kDa). To demonstrate this, T.b. gambiense cells were incubated with Alexa Fluor 594 (AF594)labelled human transferrin or AF594 labelled human C3b for 0 min at 4°C (surface binding conditions), for 2 min or 5 min at 37°C, respectively, in the presence or absence of the ISG65 and ISG75 (a paralog protein of ISG65) . Alternatively, non-labelled C3b was added at 4-fold molar excess. Cells were then fixed and subjected to flow cytometry using an excitation wavelength of 560 nm in combination with a PE-Texas Red filter. Non-treated control cells served to determine the gate for AF594 positive cells.