Facscalibur apparatus

Fetal bovine serum (FBS), f-MLP (N-Formylmethionyl-leucyl-phenylalanine), luminol, HEPES solution, RPMI 1640 medium, and L-glutamine were purchased from Sigma-Aldrich Chemie GmbH (Steinheim, Germany). Phosphate-buffered saline (PBS Ca²⁺-free) and penicillin-streptomycin were purchased from Gibco (Grand Island, USA). Lipopolysaccharide (LPS from Escherichia coli 0111:B4) was purchased from Merck (Kenilworth, USA). Pancoll human was obtained from PAN Biotech (Aidenbach, Germany). Human Quantikine ELISA Kits and propidium iodide were purchased from BD Biosciences (San Jose, USA). Dexamethasone (DEX) and quercetin (QU) (> 95% UHPLC purity) were purchased from Sigma-Aldrich GmbH (Steinheim, Germany). Acetonitrile, methanol, and formic acid for UHPLC were purchased from Merck (Darmstadt, Germany). Water for UHPLC was purified with a Millipore Simplicity System (Bedford, USA). All solvents used for chromatography were of HPLC grade. Absorbance and luminescence were measured using a BioTek microplate reader (Highland Park, USA). Flow cytometry was performed using a BD FACSCalibur apparatus (BD Biosciences, San Jose, USA). Brain-heart infusion broth (BHI) and Mueller-Hinton broth (MH cation-adjusted) were purchased from Becton Dickinson (Franklin Lakes, USA).

The cellular immune response was assessed using 10⁶ splenocytes that had been cultured in RPMI for 72 h in vitro at 37°C and 5% CO₂ in the presence or absence of 5 μg of NH36. The multiparameter analysis (51 (link), 52 (link)) was carried out to assess the intracellular production of IL-2, TNF-α, and IFN-γ cytokines by CD4⁺ and CD8⁺ T lymphocytes. For this evaluation, the cells were treated with brefeldin (SIGMA) at a final concentration of 10 μg/ml, incubated for an additional 4 h, and then stained with rat anti-mouse CD4FITC (clone GK1.5) and CD8FITC (clone 53–6.7) monoclonal antibodies (R&D systems, Inc.) and further stained with IFN-γAPC, IL-2-PerCP-Cy5.5, and TNF-αPE monoclonal antibodies (BD-Pharmingen) as described before (48 (link)). For the ICS methods, 100,000 lymphocytes were acquired using a BD FACScalibur apparatus. Data were analyzed using the Cell Quest program. The secretion of cytokines was also evaluated in the supernatants of splenocytes by an ELISA assay as previously described (48 (link)).

Female BALB/c mice, 8 weeks old, were injected intraperitoneally with daily doses 2.5 mg/Kg of IA, IH, SMIH or Glucantime during five days and euthanized on day 1, 15 and 30 after complete treatment. For evaluation of the T cell response, spleens were removed and splenocytes were incubated for 24 h in vitro with the lysate of 10⁶ stationary phase promastigotes of L. (L.) infantum chagasi (MHOM/BR/1974/PP75), with 25 μg/mL NH36, at 37°C and 5% CO₂, during 72 h for the assay of the cytokine expression in the supernatants. Additionally, splenocytes from uninfected BALB/c mice either untreated or previously treated with IA, IH, SMIH or Glucantime were incubated in vitro with 10 μg/mL of each respective drug. For flow cytometry analysis (FACS analysis), splenocytes were incubated for 24 h with the antigens or drugs, labeled with anti-CD4FITC (clone GK1.5), anti-CD8FITC (clone 53–6.7) monoclonal antibodies (R&D systems, Inc) or with rat anti-mouse CD19-PerCP-Cy5.5 and 100,000 lymphocyte counts were acquired using a BD FACScalibur apparatus. Data was analyzed using the Flow-Jo program. The secretion of cytokines was evaluated in the supernatants of splenocytes by an ELISA assay.

Apoptosis was detected with annexin V-FITC assay (BD Pharmingen, San Diego, CA, United States) according to manufacturer’s instructions. A375 cells were seeded in 35 mm culture dishes and allowed to attach overnight. The cells were treated with NAP-HBTA (100 μM) for 24-48-72 h. To detect early and late apoptosis, both adherent and floating cells were harvested together, washed twice with PBS and resuspended in annexin V binding buffer at a concentration of 10⁶ cells/mL. Subsequently, 5 μL of FITC-conjugatedAnnexin V and 5 μL of PI were added to 100 μL of the cell suspension (10⁵ cells). The cells were incubated for 20 min at room temperature in the dark and subsequently analyzed using a two-laser equipped FACSCalibur apparatus and the CellQuest analysis software (Becton Dickinson, Mountain View, CA, United States).

Mice were stimulated with intraperitoneal injection of baby hamster kidney (BHK)-570 cells (LGC Standard, ATCC®, Molsheim, France; 5.10⁶ cells in RPMI-medium, 200 μl per mice), and the production of IgM and IgG against the hamster cells was analyzed on days 3, 5, 7, and 10 postimmunization. For analysis of the IgM and IgG titer in the murine sera, 5.10⁵ BHK-570 cells were dispatched per well in a 96-well V-bottom plate and 5 μl of serum sample was added. After incubation of 30 minutes on ice, BHK-570 cells were washed two times with PBS. Secondary antibody (goat anti-mouse IgM antibody-PE or goat anti-mouse IgG antibody-FITC, Biolegend, ImTec Diagnostics N.V. Antwerp, Belgium) was added and incubated for 30 minutes on ice and protected from light. After 3 washes with PBS, IgM and IgG were assessed by flow cytometry with the 3-color Becton Dickinson FACSCalibur apparatus. Ig titer is expressed as mean fluorescence intensity (MFI).