Facs lsrii

Plasmodium falciparum parasite (3D7) were cultured in fresh red blood cells (RBCs) at 5% hematocrit with media constituted of bicarbonate buffered RPMI 1640 (RPMI 1640 Medium, Life Technologies, Grand Island, USA) supplemented with 5% albumax (AlbuMAX^® I Lipid-Rich BSA, Life Technologies, Grand Island, USA), 200 μM hypoxanthine and 20 μg/ml gentamycin as described previously48 (link). The cells were maintained at 37 °C and a gas mixture of 5% CO₂, 1% O₂ and 94% N₂. After 5 cycles of parasite growth, the mixed culture was synchronized using 5% D-sorbitol (D-sorbitol, Sigma, St. Louis, USA) treatment49 (link) and allowed to grow one more cycle then were washed to get rid of floating hemozoin. Parasites were mounted onto glass slides and stained with Giemsa (Giemsa stain, Sigma, St. Louis, USA). The parasitemia level was estimated by manual counting under microscopic observation after Giemsa staining and confirmed by staining cells with syber-green (SYBER^®Green, Life Technologies, Grand Island, USA) dye and counting with BD LSRII FACS (BD Biosciences, San Jose, USA). Appropriate dilutions were made using non-infected blood in RPMI 140 for further analysis. Optical microscopy was used to confirm the stages of parasites. It turned out that nearly 98% of parasites were in the ring stage and 2% in the early trophozoite stage in this study.

Phosphatidylserine on the cell surface was detected with Annexin V-FITC (Sigma). DNA was detected with propidium iodide (PI), (Sigma). Cells were transfected 8 hours prior staining with plasmids encoding NOA1 (wild type and ΔMTS mutant), incubated with or without 1 µM pan-caspase inhibitor Z-VAD-FMK (Sigma), detached by incubation in a trypsin/EDTA solution, washed twice with cold PBS and resuspended in 100 µl PBS, followed by incubation with 0.25 µg/ml FITC-conjugated Annexin V and 1 µg/ml PI in PBS for 15 min at room temperature in the dark. Before flow cytometry (BD LSRII FACS), cells were washed twice with 1x PBS and resuspended in 500 µl PBS. Data were analyzed using the FACS diva version 6 software.

For cytokine determinations, the supernatants from the lymphoproliferation assays were thawed and analyses were performed using the BD CBA Mouse Th1/Th2/Th17 Cytokine Kit (BD Bioscience, San Jose, CA, USA), which measures IL-2, IL-4, IL-6, IFN-γ, TNF-α and IL-17A. The samples were measured on a FACS BD LSRII and analyzed by FCAP Array 3 Software (BD Bioscience). The theoretical limits of detection for the kit Th₁/Th₂/Th₁₇ are 0.1 pg/mL for IL-2, 0.03 pg/mL for IL-4, 1.4 pg/mL for IL-6, 0.5 pg/mL for IFN-γ, 0.9 pg/mL for TNF and 0.8 pg/mL for IL-17A.
For cytokine detection in the supernatant from the lymphoproliferation assay after the mice were infected with PL, we thawed the samples and assayed with the IL-2, and IFN-γ using ELISA kits (BD OpTeia, San Diego, CA, United States). The detection limits of the assays were as follow: 3.1 pg/ml for IL-2, 31.3 pg/ml for IFN-γ as determined by the manufacturer.

The method was adapted from Tanaka et al. (15 (link)). Cells were irradiated at 1 and 2 Gy with photons or carbon ions at a dose rate of 0.5, 2, or 10 Gy/min. At 15 min (only for photons), 30 min, 1, 2, and 24 h after irradiation, cells were trypsinized, washed with PBS, and fixed in 70% ice-cold ethanol for at least 24 h. Cells were then resuspended in PBS for a wash and incubated in permeabilization buffer (20 mM HEPES, 50 mM NaCl, 3 mM MgCl₂, 300 mM sucrose, and 0.5% Triton X-100 in PBS). After two washes in PBSMB (PBS 1% milk, 0.1% BSA), cells were incubated for 2 h with gentle agitation in a primary antibody solution consisting of an antiphospho-histone-H2AX (serine139) mouse monoclonal IgG1 antibody (Millipore, Watford, UK) diluted at 1/2000 in PBSMB. Excess primary antibody was removed by washing twice in PBSMB buffer. A secondary antibody solution consisting of Alexa Fluor-488 goat-antimouse IgG antibody (Invitrogen) diluted at 1/1000 in blocking buffer was added to each sample and incubated for 20 min at room temperature. Excess secondary antibody was removed by washing twice with PBSMB. Cells were finally resuspended in PBS for flow cytometry analysis. A minimum of 10,000 cells were analyzed using a FACS-BD-LSRII.

The mCherry-EGFP-LC3-expressing lentiviral vector was kindly provided by Dr. Maria S. Soengas (Molecular Pathology Program, Centro Nacional de Investigaciones Oncologicas, Madrid, Spain). The vector contains a puromycin-resistant gene for selection of transduced mammalian cells. Lentivirus production and cancer cell transduction were done as described in [33 (link),34 (link)]. Transduced OE19 cells were selected with puromycin (1.5 mg/mL) for 4 days. The mCherry-EGFP-LC3B-expressing cells were seeded in 24-well flat-bottom plates at a density of 120,000 cells/well and on the next day treated as indicated for 24 h. The FACS experiments were performed on a FACS BD LSR II (BD Biosciences, San Jose, CA, USA) using BD FACSDiva software. The data was analyzed using FlowJo software, v10 (Ashland, OR, USA). To estimate the percentage of cells with high autophagic flux, a gate based on the DMSO control was set as described elsewhere [22 (link)].