An Assay for Quantifying Antibody-Mediated Phagocytosis of Parasite-Infected Erythrocytes

Abstract

Source: Quintana, M. d. P. et al., Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay. J. Vis. Exp. (2020)

This video demonstrates a flow cytometry-based phagocytosis assay involving labeled and opsonized Plasmodium falciparum-infected erythrocytes by monocytes. Flow cytometry identifies fluorescence in monocytes, confirming the successful identification of opsonized erythrocytes through phagocytosis.

Protocol

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines. 1. Parasite culture NOTE: Follow the local regulations for human pathogens handling. Maintain Plasmodium falciparum parasites according to the standard protocol as described before in the parasite culture medium (see Table of Materials). Keep the par…

Representative Results

Figure 1: Fc receptors expressed on the THP-1 cell surface. (A) Fcγ-receptor III/CD16 (red). (B) Fcγ-receptor II/CD32 (green). C. Fcγ-receptor I/CD64 (orange). Unlabeled cells are shown in blue. Figure 2: Phagocytosis assay flow…

Materials

96 well cell culture plates, round bottom with lid	Corning	3799	Any similar plate can be used, make sure it is compatible with the flow cytometer instrument you intend to use
AlbuMAX-II	Gibco	11021-037
AlbuMAX-II (5%)	–	–	5% AlbuMAX-II (Gibco, 11021-037), 0.2g/L hypoxanthine (Sigma, H9377) in RPMI1640 (Sigma, R5886)
Anti-Red Blood Cells antibody	Abcam	ab34858	Prepare aliquots and freeze a -20°C. Use one aliquot per experiment.
DPBS	Sigma	P8622
Dynabeads Protein G	Invitrogen	10003D
Ethidium bromide solution	Sigma	E1510	Prepare a stock solution at 0.1mg/mL in RPMI1640 (R5886). Store protected from light
FC500 flow cytometer	Beckman Coulter		Any flow cytometer supporting 96 well plate format and having the appropriate lasers/filters to measure EtBr fluorescence can be used.
Fetal Bovine Serum (FBS)	Gibco	10099-141	Heat inactivate before use.
FITC mouse anti-human CD16	BD Biosciences	555406 or 556618
FITC mouse anti-human CD32	BD Biosciences	552883
FITC mouse anti-human CD64	BD Biosciences	555527
FlowLogic software	Inivai technologies		Any flow cytometry analysis can be used, for example FlowJo or Winlist
Gentamicin (10mg/mL)	Sigma	G1272
Hypoxanthine	Sigma	H9377
L-glutamine (200mM)	Sigma	G7513
Lysing solution	–	–	15mM NH4Cl, 10mM NaHCO3, 1mM EDTA
MACS CS-column and accesories	Miltenyi Biotec	130-041-305
Parasite culture medium	–	–	2mM L-glutamine (Sigma, G7513), 50µg/mL Gentamicin (Sigma, G1272), 0.5% AlbuMAX-II (AlbuMAX-II 5%) in RPMI1640 (Sigma, R5886)
Penicillin/Streptomycin (10000U and 10mg/mL)	Sigma	P0781
RPMI-1640 medium	Sigma	R5886
THP-1 culture medium	–	–	10%FBS (Gibco, 10099-141), 2mM L-glutamine (Sigma, G7513), 100U/mL Penicillin, 0.1mg/mL Streptomycin (Sigma, P0781) in RPMI1640 (Sigma, R5886)
Vario MACS magnet	Miltenyi Biotec