7 aminoactinomycin d 7aad live dead dye

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).

For uptake experiments, rested MoDCs and MoMϕs were stimulated for 1 hour with recCSP in the concentrations described or a 1:1 sporozoite:cell number of (opsonized) fluorescent Pb parasites. Opsonization of parasites was performed as described above. Alternatively, SPZ were fixed for 15 minutes at room temperature using 4% formaldehyde, which was followed by three washes with phosphate buffered saline (PBS). RecCSP had been fluorescently labelled using a commercial antibody labeling kit (ALEXA Fluor 660 antibody labeling kit, Thermo Fisher Scientific, Waltham, MA, USA) according to their protocol. After 1 hour of stimulation, cells were harvested, stained with 7-aminoactinomycin D (7-AAD) live/dead dye (Invitrogen, Waltham, MA, USA) 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).