Cd209 dcn46

In addition, the phenotype of MoMɸs was assessed by flow cytometry and cytokine measurements. For this purpose, MoMɸs were stimulated with 1:1 ratio SPZ, salivary gland extract (SGE) as a negative control, medium as background or 100 ng/mL lipopolysaccharide (LPS) as a positive control for 24 h at 37 °C + 5% CO₂. After 24 h supernatant was collected and the cells were harvested, washed with PBS and stained for CD209 (DCN46; BD Bioscience), CD163 (9332, BD Bioscience), PD-L1 (MIH1; BD Bioscience) ILT3 (zm4.1; Biolegend), CD206 (15-2; Biolegend), CD80 (L307.3; BD Bioscience), CD25 (2A3; BD Bioscience), aqua live/dead staining (Thermofisher), Fc-block (BD Bioscience) in PBS for 30 min at 4 °C and measured by flow cytometry using LSR Fortessa (BD bioscience, San Jose, CA, USA). The data was analysed by FlowJo version 10.8 (FlowJo LLC, Ashland, OR, USA). After 24 h stimulation of MoMɸs with SPZ from day 14, 17 and 20 post blood meal, and controls (LPS, SGE, medium) the supernatants were diluted 1:5 (IL6) or 1:2 (IL10 and TNF) and analysed by standard ELISA (Sanquin, The Netherlands).

On day 7, rested immature MoDCs and M0 MoMϕ were exposed for 24 hours to recombinant pf circumsporozoite protein (recCSP; Alpha Diagnostics, San Antonio, TX, USA catalognr: CSPF17-R-10), 20.000 Plasmodium falciparum (Pf) SPZ, a matched volume of salivary gland extract (SGE) of uninfected control mosquitoes or medium as a true negative control and ultrapure lipopolysaccharide (LPS; 100ng/ml; Escherichia coli 0111 B4 strain, InvivoGen, San Diego, CA, USA) as an inflammatory control. When different well sizes were used, the same 1:5 sporozoite:cell ratio was maintained. Importantly, SGE was used as a matched control as SPZ preparations do contain traces of salivary gland material. After 24 hour, cells were harvested, stained with 7-aminoactinomycin D (7AAD) live/dead dye (Invitrogen, Waltham, MA, USA), and antibodies against CD80 (L307.4; BD Biosciences), CD86 (N331 FUN-1; BD Biosciences), CD25 (2A3; BD Biosciences), CD197 (3D12; BD Biosciences), CD206 (15–2; Biolegend), CD209 (DCN46; BD Biosciences), PD-L1 (MIH1; BD Biosciences), PD-L2 (MIH18; eBiosience) and Immunoglobulin-like transcript 3 (ILT3; zm4.1; Biolegend) and analyzed by Flow Cytometry using a BD LSR Fortessa (BD Biosciences, San Jose, CA, USA) and analyzed in FlowJo version 9.9.6 (FlowJo LLC, Ashland, OR, USA).