Tcs sp5 x confocal microscope

Confocal images were taken at the Center of Excellence for Nanoscience and Nanotechnology (Department of Biochemistry, Molecular and Structural Biology, Jožef Stefan Institute, Ljubljana, Slovenia) or at the Microscopy Service of the Autonomous University of Barcelona (Edifici C Campus UAB, Cerdanyola del Vallés, Barcelona, Spain). Leica TCS SP5 X confocal microscope (Leica Microsystems GmbH, Wetzlar, Germany) was used for optical slicing (oil objective ×60, NA = 0.5). Proteostat was excited at 500 nm with white light laser (WLL) and DAPI was excited at 405 nm with an ultraviolet (UV) laser. For the BiFC experiments, YFP was excited with an Argon multilineal laser at 488 nm in live adherent cells. The emission signal was followed with a photomultiplier detector (PMT) or a hybrid detector (HyD). Leica LAS AF software (Leica Microsystems GmbH, Wetzlar, Germany) was used for image analysis.

CD4 T cells were purified from spleen by negative selection using biotinylated anti-CD8, anti-B220, anti-Gr-1, anti-F4/80 and anti-Ter119 antibodies and anti-biotin microbeads following the manufacturer´s instructions (MACS Miltenyi Biotec). Bone marrow derived, LPS matured dendritic cells were loaded with 1 µg/ml MOG35-55 peptide for 2 hr at a concentration of 10⁶ cell/ml. 1 × 10⁵ loaded DCs were mixed with 5 × 10⁵ CD4 T cells in 200 µl R10 medium and seeded on poly-L-Lysine coated glass-bottom dishes (Mattek). Cells were fixed after 30 min by adding 200 µl warm 6% PFA in R10 medium for 20 min at 37°C. After permeabilization with 0.2% Triton X-100 in PBS for 15 min and blocking with 1% BSA for 1 h cells were stained with antibodies against LFA-1 (clone 2D7, BD Biosciences) and p-Tyrosine (PY199, Santa Cruz Biotechnology). Phalloidin Alexa350 was used to visualize actin. Cells were imaged with a Leica TCS SP5 X confocal microscope (Leica Microsystems) equipped with a 63 × NA 1.40 oil objective and Leica Confocal Software (LAS AF). Single channels were imaged sequentially. All pictures were processed with Photoshop (Adobe Systems, San José, California, USA).

Cells were cultured on coverslips, fixed in 4% paraformaldehyde for 15 min at room temperature, then treated with 0.1 M TRIS-glycine (pH 7.4) for 15 min, permeabilized with 0.3% TritonX-100 for 15 min, blocked with 5–10% BSA for 30 min and incubated overnight at 4 °C with primary antibody against gastric HKα1 (Calbiochem 119101, RRID:AB_211408, 1:100), non-gastric HKα2 (1:300, C384-M79) and HKβ (1:100, A274, Sigma). Samples were incubated with appropriate secondary antibodies conjugated to Alexa 488 (Invitrogen, 1:400). DAPI (1:400, Molecular probes) was used as nuclear staining. Fluorescence was examined with 40× 1.3 NA or 63× 1.2 NA objectives in Leica TCS SP5 X confocal microscope (Leica Microsystems, Heidelberg). Images were analyzed in Leica software.

PANC-1, PSCs and HEK-293 cells have been transfected with 2–3 μg/ml of the construct encoding for P2X7R variants. The vector used in this study was pcDNA1.3 + and the hP2X7R gene is flanked by the Nhel and EcoRV restriction sites. The WT gene and P2X7R gene contain His155. The plasmid with the gene and with/without the mutation of interest was separated by an IRES sequence from GFP sequence, and therefore P2X7R and GFP were expressed simultaneously but separated. Thus, the cells containing and expressing the vector were GFP-positive. Transient transfection has been performed before each experiment using FuGENE HD Transfection reagent (Promega, E2311), ratio 1:3. We used green fluorescence to distinguish the transfected (P2X7R + GFP) from non-transfected cells expressing the native receptor. Overall cell morphology was assessed by imaging of cells (λ_ex = 588 nm, λ_em = 510 nm) in Leica TCS SP5 X confocal microscope (Leica Microsystems, Heidelberg, DE). The expression of the constructs was evaluated by Western blot. Primary antibodies were against the P2X7R (APR-004, Alomone Labs, Jerusalem, IL, RRID: AB_2040068), GFP (SC-8334, Santa Cruz, Tilst, DK, RRID:AB_641123), β-Actin (Sc-47778, Santa Cruz, Tilst, DK, RRID: AB_626632) and secondary antibody was HRP-conjugated (EZ-ECL-Biological Industries, Fredensborg, DK) and visualized with Fusion FX (Vilber Lourmat, Eberhardzell, DE).

Polarized T84 cells were incubated with GC apically for 6h. Then the cells were incubated with the CellMask dye (5μg/ml, Life Technologies) in the basolateral chamber only for 15 min, and xz images were acquired using Leica TCS SP5 X confocal microscope (Leica Microsystems, Buffalo Grove, IL). The number of epithelial cells displaying CellMask staining at the apical membrane was countered visually as the percentage of the total number of epithelial cells in each randomly acquired image.

S. aureus was labeled with Calcein Blue, AM (5 μM, Thermo Fisher Scientific) as previously described (Cohen et al., 2016 ). BMDMs or primary human monocytes were treated as described above. In select experiments, mitochondria were labeled with Mitotracker CMXRos (Molecular Probes) prior to incubation with bacteria. The cells were incubated with labeled S. aureus at MOI 1 in a 96-well plate. For caspase-1 imaging in cells, FAM-FLICA (Immunochemistry Technologies) was added to each well 1 hr post bacterial inoculation, and cells were stained in accordance with the manufacturer’s instructions. The cells were washed 3 times in fresh PBS prior to imaging, and 20 μL cell suspension was loaded onto a coverslip. The cells were maintained at 37°C and 5% CO₂ throughout the imaging process. To image ASC, cells were allowed to adhere to coverslips overnight, prior to labeling mitochondria and infecting as described above. Following a 1-hr incubation with bacteria, cells were fixed with 10% formalin (10 min), permeabilized with 0.5% Triton X-100 in PBS (10 min), blocked with BlockAid (Thermo Fisher Scientific, 1 hr), and stained overnight with anti-ASC/TMS1 (Novus Biologicals). ASC/TSM1 was visualized with AF647 conjugated anti-rabbit IgG secondary (Molecular Probes). The slides were imaged using a Leica TCS SP5 X confocal microscope (Leica Microsystems).

T84 cells were seeded at 1×10⁵ per transwell on the underside of transwells [88 (link)] and cultured for ~10 days until TEER reached >1000 Ω. Cells were pre-treated with or without the Ca²⁺ inhibitors, 2APB (10 μM, Sigma, Saint Louis, MO) and BAPTA (50 μM, Sigma), for 1 h and incubated with GC (MOI of 10) apically in the presence or absence of the inhibitors for 4 h. Then cells were incubated with the fluorescent Ca²⁺ indicator Fluoforte (100 μg/ml, Enzo Life Sciences, Farmingdale, NY) or Fluo-4 (100 μM, Life Technologies) for 1 h. Confocal xz images were acquired in the presence of the membrane dye CellMask (5 mg/ml, Life Technology) using Leica TCS SP5X confocal microscope (Leica Microsystems, Buffalo Grove, IL), based on the instruction by manufacturers. To quantify the intracellular Ca²⁺ level, the cytoplasmic region of individual cells was manually selected based on the CellMask staining in randomly acquired confocal images, and the mean fluorescent intensity (MFI) of Fluoforte and Fluo-4 in the cytoplasmic region was measured using the NIH ImageJ software.