S1 100ml

Peptide arrays were blocked with phosphate-buffered saline (PBS) + 3% bovine serum albumin (BSA) (PBSB) ON at 4°C or for 2h at room temperature (RT), followed by three 5 minute washes of PBS + 0.05% Tween 20 (PBST). In order to assess the peptide recognition by sera from VL patients and NEHCs, separate arrays were hybridised with pooled Sudanese serum samples positive for VL and with commercial pooled NEHC sera (S1-100ML, EMD Millipore Corporation, USA), respectively, diluted in PBST + 3% BSA (PBSTB), for 1h. After five 5 minute washes with PBST, they were incubated with fluorescent mouse anti-Human IgG1 Fc—Alexa Fluor 488 antibody (AB) (A-10631, ThermoFisher) diluted 1:1000 in PBSTB, for 1h at RT. Followed by five 5 minute washes with PBST, the arrays were incubated at 50°C until completely dry.
Images were acquired at 500ms and 20 dB gain with a digital CCD camera (ORCA-R², Hamamatsu, Japan) coupled to a fluorescence microscope (model BX53, Olympus, Germany) equipped with filter cube U-FGFP (N271350, Olympus, Germany). The fluorescence of the spots was quantified using the software cellSens Dimensions v.1.7 (Olympus GmbH, Germany)

Cells were washed once with sterile PBS and incubated with 500 μl DMEM on the apical surface. On the basal surface, cells were treated with 500 μl DMEM containing the C5b-6 complex (0.2 mg/ml) and C7 (60 μg/ml), C8 (50 μg/ml), and C9 (60 μg/ml) purified complement proteins. Cells were incubated for 1, 4, 8, 24, and 48 h at 37°C/5% CO₂, unless stated otherwise. As a control, C9 was omitted; thus, cells were treated with C5b-6 complex (0.2 mg/ml) and C7 (60 μg/ml) and C8 (50 μg/ml) purified complement proteins only. Purified complement component proteins referenced above were purchased from Complement Technology, unless stated otherwise. Normal human serum was purchased from Merck Millipore (S1-100ML), and heat inactivation was carried out at 56°C for 1 h.