4.6. Evaluating Protein-Cell Interaction by Flow Cytometry

rPvMSP10, rPvMSP10-N, rPvMSP10-C, rPvRBSA and Trx binding to human reticulocytes was evaluated in triplicate by flow cytometry. 10⁶ CD71+ enriched cells were incubated with different µM concentrations of each protein (0 to 5 µM) at 4 rpm for 16 h at 4 °C. Binding competition assays involved pre-incubating the cells with EGF-like domain-derived peptides located in PvMSP10-C: peptide 1 (42418) in ³⁶⁸NHICEYSKCGANARCYIVEK³⁸⁷, peptide 2 (42419) in ³⁸⁸DKEECRCRANYMPDDSVDYF⁴⁰⁷, peptide 3 (42420) in ⁴¹⁵KDCSKENGNCDVNAECSIDK⁴³⁴ and peptide 4 (42421) in ⁴³⁵NKDIKCQCKFNYIGDGIFCV⁴⁵⁴); they were then synthesised and purified (Supplementary Figure S6) as previously described [50]) in a 1:100 (protein:peptide) µM ratio at 4 °C for 1 h at 4 rpm. Mycobacterium tuberculosis (peptide 5 (39266): APSNETLVKTFSPGEQVTTY) and PvRBSA (peptide 6 (40893): TASSESLAESNDAPSNSYESY) [7] -derived peptides were used as negative controls (all cysteine residues were replaced by serine to avoid peptide polymerisation). The samples were washed 3 times with PBS and then incubated with monoclonal Ab Anti-his tag APC conjugated mouse IgG1 clone AD1.1.10 diluted 1:50 (R&D systems, Minneapolis, MN, USA) and Retic-Count for 20 min in the dark. Protein-cell interaction was determined by analysing 100,000 events on a FACSCanto II cell analyser, using the aforementioned parameters. A gating strategy was used for selecting an RBC homogeneous population using FlowJoV10 software; reticulocyte binding percentage was analysed by plotting APC against FITC signals (Supplementary Figure S5). Average rPvMSP10 binding to human adult reticulocyte percentage was considered 100%; all data obtained with individual peptides were compared to this positive control.