24 well flat bottom plates

Spleens and LNs removed from 12/36 BALB/c mice, 10 weeks after inoculation of L. donovani-SL (n = 6/12) and L. donovani-1S (n = 6/12), were used to extract cells for in vitro stimulation. Cells were extracted from freshly removed organs and stimulated with soluble parasite antigens.
Splenocytes and lymphocytes were separated into single-cell suspension by passing through a fine wire mesh. The red blood cells were lysed with ACKlysis buffer. Splenocytes and lymphocytes were then re-suspended in culture at 3 × 10⁶ cells mL⁻¹ in complete Dulbecco's modified Eagle's medium (DMEM) supplemented with 20 mm 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) HEPES, 10% heat-inactivated FBS, 20 U mL⁻¹ penicillin, 20 µg mL⁻¹ streptomycin and 50 µmβ-mercapto-ethanol (Sigma) at 37 °C in 5% CO₂ using 24-well, flat bottom plates (Nunc, USA). Cells were stimulated with SLA at a concentration of 100 µg well⁻¹ and incubated at 37 °C for 72 h, prior to collection of culture supernatants for cytokine assays (Selvapandiyan et al.2009 ).

For Ad5 titration, 293 cells were seeded at cells per well in 24-well flat bottom plates (Nunc, Denmark). After 24 h, the cells from three wells were trypsinized and the cell concentration was determined. The cell culture medium was removed from the remaining wells and replaced with 1 mL of viral suspensions ( – ) diluted in fresh medium. After 17 to 20 hours, the cells were collected in Dulbecco's phosphate-buffered saline (D-PBS, Gibco, UK) with 5% FBS and immediately analyzed by flow cytometry (CyFlow space, Partec GmbH, Germany). Both the initial feedstock and the samples collected during the 10-fold concentration steps were analyzed in the same assay, thus using the same cell culture, to eliminate assay-to-assay variability. The infectious particle (IP) recovery, , was calculated as follows:

where is the initial infectious particle concentration and is the value at the end of the 10-fold concentration step; represents the initial volume and the final volume obtained after the 10-fold concentration step.

Heparinized blood samples were collected from all recruited individuals. The survey was first approved by the Research with Human Beings Ethics Committee of the Federal University of Rio Grande do Norte (protocol CAAE: 56191416.0.0000.5537). PBMCs were separated by centrifugation over a gradient of Ficoll-Hypaque (Merk, São Paulo, Brazil). Mononuclear cells were resuspended in RPMI supplemented with 10% human AB Rh-serum (Merk, São Paulo, Brazil), 10 mM HEPES (Merk, São Paulo, Brazil), 1.5 µM L-glutamine (Merk, São Paulo, Brazil), 200 IU of penicillin per ml, and 200 µg of streptomycin per ml (Merk, São Paulo, Brazil) and were adjusted to 2 × 10⁶ cells/mL. PBMCs (10⁶ cells per well) were cultured in vitro in 24-well flat bottom plates (Nunc, Roskilde, Denmark) at 37 °C in a humidified atmosphere of 5% CO₂ in air and in the presence of different concentrations of blank-cationic nanoparticles or medium alone. After 3 days in culture, the stimulated cells were harvested and processed for apoptosis analyses. For apoptosis analyses, FITC Annexin V apoptosis detection kits (BD) were used following the manufacturer’s instructions. Twenty thousand events were acquired from each sample into a lymphocyte gate using a FACS Canto II flow cytometer (BD, Canto II flow cytometer, Becton Dickinson Bioscience, San Jose, CA, USA). Annexin V and Pi were analyzed using the FlowJo vX software.

Vγ9Vδ2 T cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 (Invitrogen) supplemented with 10% heat-inactivated AB serum, 2 mM glutamine (Invitrogen), and penicillin–streptomycin (Sigma-Aldrich). Briefly, 1 × 10⁶ Vγ9Vδ2 T cells were seeded in triplicate sets in 24-well flat bottom plates (Nunc) and were treated in various combinations: unstimulated Vγ9Vδ2 T cells, Vγ9Vδ2 T cells stimulated with 50 IU/ml rIL2 (Peprotech), 50 IU/ml rIL2 + HDMAPP (1 nM; Echelon), 50 IU/ml rIL2 + IPP (40 μM; Sigma-Aldrich), 50 IU/ml rIL2 + plate-bound anti-CD3 monoclonal antibody (clone OKT3; BD Biosciences, USA), rIL2 + HDMAPP + γ-secretase inhibitor-X, L-685,458 (GSI-X, 15 μM) (Calbiochem, La Jolla, CA, USA), rIL2 + IPP + GSI-X, and rIL2 + anti-CD3 + GSI-X, using previously standardized concentrations. After 72 h, the viability of Vγ9Vδ2 T cells was determined by Trypan Blue cell exclusion assay. The viability ranged from 86% to 90% for untreated Vγ9Vδ2 T cells and from 93.4% to 94.8% for all other treatments previously mentioned. The harvested cells were snap-frozen in TriZol (Invitrogen) and stored at −80°C for library preparation.

To detect the influence of NOD2 on apoptosis and the signal pathway, DSCs (2×10⁵/well) were cultured in 24-well flat-bottom plates (Nunc, Penfield, NY, USA) for 48 hrs with or without MDP. The attached DSCs were harvested by using 0.25% typsin (without EDTA). The DSCs in suspension (individual Falcon 2054 polystyrene round-bottom tubes, Becton Dickinson, Franklin Lakes, NJ, USA) were washed with DMEM/F12 medium, resuspended in 200 µl DMEM/F12 (without FBS) using the Annexin V-Biotin Apoptosis Detection Kit (Calbiochem, Merck KGaA, Germany). In parallel, isotypic IgG antibodies were used as controls. Samples were analyzed by FACS Calibur flowcytometer (Becton Dickinson) using Cell Quest software (BectonDickinson). Statistical analysis was conducted by using isotype matched controls as the reference.

Metabolic activity was analyzed by quantification and control-normalization of fluorescent resorufin 4 h after addition of resazurin (final concentration 100 µM; Alfa Aesar, Karlsruhe, Germany) at λ_ex = 535 nm and λ_em = 590 nm using a multimode reader (Tecan, Männedorf, Switzerland). For this assay, 1 × 10⁴ PDA cells were seeded one day before plasma treatment. As a control, the ROS scavenger n-acetylcysteine (NAC, final concentration: 2 mM; Sigma, Taufkirchen, Germany) was used. In a different setup, 7.5 × 10⁴ PDA cells were seeded in flat-bottom 24-well plates (NUNC, Roskilde, Denmark). One hour after plasma treatment, transwells were added to each well, and 7.5 × 10⁴ RAW cells were added into the transwell. Twenty-four hours later, the transwells were removed, and resazurin was added and analyzed as described above. For analysis of cell viability, 2 × 10³ PDA cells were added to each well of a 96-well plate, and propidium iodide (PI, final concentration 1 µg/mL; Merck, Darmstadt, Germany) was added. The cells were imaged (brightfield and PI) kinetically, and the number of viable cells against all cells was counted manually for each time point.