7 aad staining

The in vitro cytotoxicity assay was performed by flow cytometry using 7-AAD staining (Invitrogen, Life Technologies) as previously reported [17 (link),18 (link)]. BMDMs-infected 24 hours with L. donovani were used as target cells. Nonadherent splenocytes from one-month L. donovani-infected mice were stimulated with SLA (10μg/ml) and IL-2 (20ng/ml) for 3 days, after which they were recovered and used in the cytotoxic assay. Briefly, effector cells were incubated for 48 hours with previously L. donovani-infected BMDMs at different E:T cell ratios (2:1; 5:1 and 10:1), in the presence of normal levels of L-glutamine (2mM; CTRL), high levels of glutamine (5 mM; + L-glut) and without glutamine (- L-glut).

Flow cytometry analysis was done as described earlier^{6 (link)}. Cells (1 × 10⁵) obtained after enzymatic dissociation of 1° and 2° cervicospheres were stained with anti-CD49f-FITC (GoH3; BD-Pharmingen), anti-CD71-APC (M-A712; BD-Pharmingen), and/or anti-CD133-PE for 60 minutes at 4 °C in staining buffer (2% BSA in 185 PBS). Corresponding isotypes were used as control. Cells were washed in PBS, centrifuged, and finally resuspended in 300 µL analysis buffer (1% BSA/2 mmol/L EDTA in PBS). FACS sorting was done as described earlier^{6 (link)}. In brief, 1 × 10⁶ cells were incubated with anti-CD49f-FITC, anti-CD71-APC, and/or anti-CD133-PE, and were finally resuspended in 500 µL analysis buffer. The dead cells and debris were excluded after 7-AAD staining (Invitrogen). Data were collected on FACSAriaIII cell sorter (BD Biosciences) and analyzed using FlowJo software (TreeStar).

Harvested tumors were manually minced into small pieces with scalpels before incubating with 350 μg/mL Liberase TL (Roche) for 20 min at 37 °C and filtered through a 70-µm cell strainers (BD Biosciences, Bedford, MA, USA) to obtain single cell suspension. The cells were subjected to Ammonium-Chloride-Potassium (ACK) lysis (5 min) before staining with 10% normal mouse serum and anti-mouse CD16/CD32 antibody (clone 2.4G2) to block Fc receptor for IgG (FcγRs). Single-cell suspensions of tumor-infiltrating lymphocytes were stained using the following antibodies: CD8α (clone 53-6.7), CD4 (clone L3T4), CD3ε (clone 145-2c11), CD11b (clone M1/70), F4/80 (clone BM8), CD45.2 (clone 104), Ly6G (clone 1A8), PD-L1 (clone MIH5). LAG-3 (C9B7W), and CTLA-4 (9H10). Then, 7-AAD staining (Invitrogen, Carlsbad, CA, USA) was used to exclude dead cells. All stained cells were analyzed on a LSRII cytometer (BD) and data analysis was performed with FlowJo Software v10 (Tree Star, San Carlos, CA, USA).

Cell supernatants were collected at different time points following transfection. Fresh media was added to the cells after collecting the samples. 50 μl of the supernatants were mixed with 50 μl of the Metridia luciferase substrate (coelenterazine, Invivogen, France). The luciferase activity was determined by measuring emitted light with a luminometer (FLUOstar OPTIMA, BMG LABTECH, Germany). Luciferase activity is expressed in relative light units (RLU).
To determine the number of transfected cells EGFP expression was evaluated by means of fluorescence microscopy (Zeiss Axiovert 200 M, Carl Zeiss Microscopy GmbH, Germany) as well as by flow cytometry (FACSCanto, BD Bioscience, Germany) 24 h post transfection. For flow cytometry analysis, the cells were washed with PBS, detached by exposure to trypsin and suspended in a flow buffer (PBS with 2% FCS, 2mM EDTA, 0,005% NaN₃; Sigma Aldrich, Munich, Germany). Dead cells were excluded based on 7-AAD staining (eBioscience, Germany). 10 000 live cells per sample were analysed. Data were analysed with FCS Express 4 Flow Cytometry software (De Novo Software, USA).

Human PDL cells were grown in two T25 dishes (Greiner Bio One, Monroe, NC, USA) to confluency. The cells were then trypsinized and resuspended in flow cytometry buffer (eBioscience, San Diego, CA, USA). These cells were subsequently blocked for non-specific interactions using human Fc receptor binding inhibitor (eBioscience, San Diego, CA, USA) at room temperature. For surface staining we used the following antibodies: rat mAb CD44-FITC (1:50 dilution, ab19622, Abcam, Cambridge, MA, USA), anti-CD146 (1:40 dilution, 50–1469, eBioscience, San Diego, CA, USA) and anti-human CD31 (1:40 dilution, 11–0319, eBioscience, San Diego, CA, USA), and incubated for 30 min in the dark at 2–8°C. The cells were washed three times with flow cytometry buffer after centrifugation at 400–600xg for 5 min. After the final wash, the cells were suspended in flow cytometry buffer and 7-AAD staining (eBioscience, San Diego, CA, USA), before being fixed with intracellular fixation buffer (eBioscience, San Diego, CA, USA). The tubes were then analyzed using a LSRII cytometer equipped with 488 nm, 561 nm, 640 nm, 405 nm and 350 nm lasers and all flow cytometry data were analyzed with Flow Jo software. Side and forward scatter of aggregates in cell lysates were determined using log scale plots. Voltage settings for the PerCp-A, FITC and efluoro660 channels were kept constant for all experiments described.