Eclipse ti e inverted microscope

RBECs were cultured until confluence in 12 well plates and treated with TGF-β1 or ACM for 5 h. B16/F10 melanoma cells (2 x 10⁴/well) were plated onto the monolayer. For inhibitor studies, cells were preincubated with SB-431542 for 60 minutes. Cells were monitored over 10 h and phase contrast images were taken using an Andor NEO sCMOS camera (Andor Technology, Belfast, UK) connected to the Nikon Eclipse Ti-E inverted microscope equipped with a home built incubator set to 37°C. Photographs were made every 5 min and transmigrated cells were counted.

Cells grown in Lab-Tek™ (Nunc) were washed with HCSS buffer (120 mM NaCl, 5.4 mM KCl, 0.8 mM MgCl₂, 15 mM glucose, and 20 mM Hepes [pH 7.4]) and incubated with 2.5 µM Fura-2-AM (Life Technologies) for 30 min at room temperature in the dark. Cells were washed with HCSS and incubated 15 min at room temperature to allow Fura-2 de-esterification. Images were recorded with a Nikon Eclipse Ti-E inverted microscope, with excitation of Fura-2-AM loaded cells alternately at 340 and 380 nm. Emission at 510 nm was recorded. Conversion of Fura-2 ratios into cytosolic calcium concentrations was performed as previously described^{51 (link)}.

Primary cilia consist of acetylated microtubule structures and were measured by direct immunofluorescence with anti-Acetylated α-tubulin staining in the absence or presence of 20-hour incubation with 1μM rapamycin. The cells were fixed for 10 minutes (4% paraformaldehyde/2% sucrose in PBS) and permeabilized for 5 minutes (10% triton X-100). Acetylated α-tubulin (1:10,000 dilution, Sigma Aldrich, St. Louis, MO) and fluorescein isothiocyanate (FITC)-conjugated (1:1000 dilution, Vector Labs Burlingame, CA) antibodies were each incubated with the cells for 1 hour at 37°C. Slides were then mounted with DAPI (Southern Biotech, Birmingham, AL) hard set mounting media. Nikon Eclipse Ti-E inverted microscope with NIS-Elements imaging software (version 4.30) was used to capture images of primary cilia. Automated image acquisition was conducted in 100X magnification fields and Z-stacks (0.1 μm slices) to create a large 3D image. This was done to select cilia from a confluent monolayer since it mimics the physiological structure of the epithelial and endothelial cells.

We employ a fully automated imaging system built around a Nikon Eclipse Ti-E inverted microscope using a Nikon Plan Apo \lambda 40× 0.95 N.A. dry objective and Grasshopper3 GS3-U3-23S6M-C camera from Point Grey Research. For bright field imaging a halogen lamp light source is used. For epi-fluorescence imaging we use a metal-halide lamp source and two fluorescence filter sets, violet (Semrock Inc FF01-390/40 excitation filter, FF416-Di01 dichroic filter, FF01-460/60 emission filter) and GFP (Semrock Inc. FF01-472/30 excitation filter, FF495-Di03 dichroic filter, FF01-520/35 emission filter).
For the experiment, three micro-well (50 μm by 50 μm; Microsurfaces)-containing imaging petri dishes were loaded with GEN4P and GEN4N, respectively. The dishes were placed in a custom-built environmental chamber at 37 °C, 5 % CO₂ and 100 % humidity. After registering 50–100 single micro-well positions per each type of cell, the cell positions were imaged in bright field, violet and GFP channels for 20–40 h so that it is possible to observe one complete cell cycle.
A MATLAB program developed in house was employed to extract the data from frames and the division time between mother and daughter cells was measured and tabulated (Fig. 1).

NSC‐34 cells plated on glass coverslips were fixed with 3% paraformaldehyde and 0.1% glutaraldehyde in phosphate‐buffered saline (PBS) for 10 min at 20°C. After washing with PBS, samples were quenched by incubation with 50 mM NaBH₄ in PBS for 7 min, washed in PBS and then permeabilized for 30 min in PBS containing 0.2% Triton X‐100 and 3% bovine serum albumin (BSA). Following blocking with 3% BSA in PBS, samples were incubated with primary antibodies diluted in blocking solution, washed with PBS and incubated with goat anti‐rabbit or anti‐mouse secondary Igs conjugated to AlexaFluor 546 or AlexaFluor 633 (1:1,000) (Invitrogen). Following final washings in PBS, the samples were mounted in Mowiol‐DABCO mounting medium containing 10% (w/v) Mowiol 4‐88 (Calbiochem), 25% (w/v) glycerol and 2.5% (w/v) DABCO (1,4‐diazobicyclo[2.2.2]octane) in 100 mM Tris–HCl pH 8.5. Samples were analysed on a Nikon Eclipse Ti‐E inverted microscope equipped with a Nikon N‐SIM Super Resolution System. Confocal images were captured using a CFI Plan Apo IR SR 60X water immersion objective and then reconstructed using Nikon Imaging Software Elements AR with N‐SIM module. ER–mitochondria interactions were quantified by Mander's coefficient using ImageJ software. Co‐localized signals were displayed using the ImageJ 1.44p RG2B co‐localization plug‐in to determine co‐localized pixels.

Ex vivo thrombus growth analysis was conducted as described previously [46 ,66 (link)]. Briefly, hirudinated whole blood was preincubated with DiOC-6 (500 nM) and perfused through the parallel plate flow chambers with fibrillary collagen (100 µg/mL) covered glass at 100 s^-1 shear rate. Blood with “low platelet count” was obtained after the RBC settling for 1h, LRP collection, and supplementation of 80% of LRP by Tyrode’s calcium buffer with hirudin. Diluted LRP was then reintroduced to the whole blood and incubated for 30 min. Comparison of Tyrode’s supplemented blood and platelet free plasma supplemented blood did not provide any significant differences (data not shown). Thrombus growth was observed using a Nikon Eclipse Ti-E inverted microscope (Tokyo, Japan). Ten fields of view (FOV) were monitored during the first 10 min of the experiment and then, 10 different FOVs were monitored during the following 10 min of the experiment. Polymorphonuclear leukocytes (PMNs) were determined by diffuse DiOC-6 labeling, distinctive from typical thrombi labeling (see Figure S5).

Samples of GUVs and SaroL-1 were imaged using a confocal fluorescence microscope (Nikon Eclipse Ti-E inverted microscope equipped with Nikon A1R confocal laser scanning system, 60x oil immersion objective, NA = 1.49 and four laser lines: 405 nm, 488 nm, 561 nm, and 640 nm). Image acquisition and processing were made using the software NIS-Elements (version 4.5, Nikon) and open-source Fiji software (https://imagej.net/software/fiji/).