Tcs sp8 aobs confocal microscope

For live imaging, MIA PaCa-2 cells, plated in a µ-Slide 8 Well IbiTreat (Ibidi, Gräfelfing, Germany), were transiently transfected with the fluorescent-tagged cDNA pEGFPN1 LifeAct (pEGFP-C1 Lifeact-EGFP was a gift from Dyche Mullins (Addgene plasmid # 58,470; http://n2t.net/addgene:58470; accessed on 21 December 2021, RRID:Addgene_58470). Cells were analyzed by using a Leica TCS SP8 AOBS confocal microscope equipped with 2 HyD, PL APO 63×/1.4, NA immersion objective was employed. Images of 512 × 512 pixels were acquired at pixel size = 161.51–182.01 nm. Image acquisition was performed by adopting a laser power, gain, and offset settings that allowed maintaining pixel intensities (gray scale) within the 0–255 range and hence avoiding saturation. Movies to image the actin filopodia were acquired for 5 min, taking one frame every 2.5 s. In particular, we quantified the physical parameters (number, length, and density) of filopodia using the Fiji plugin Filoquant, as described in [10 (link)].
Filopodia dynamics, in terms of velocity and tip distance, were manually tracked using Fiji plugin TrackMate. We tracked from 5 to 10 filopodia/cell over the filopodia lifetime in at least 11 cells (up to 14).

Cells were fixed in paraformaldehyde 4% for 20 min. After paraformaldehyde quenching (NH₄Cl 50 mM), permeabilization (Triton X-100 0.2% in PBS) and blocking (bovine serum albumin 3% in PBS) steps, cells were incubated 1 h in primary antibodies (rabbit anti-EEA1 primary antibody C45B10, Cell Signaling, 1/500; mouse anti-MAP 2 antibody, MAB3418, Millipore, 1/500; mouse anti-Phosphatidylinositol-3-phosphate (PI (3) P) antibody, Z-P003, Echelon Biosciences, 1/200). Cells were rinsed and incubated in anti-rabbit AlexaFluor-488 and anti-mouse AlexaFluor-568 secondary antibodies for 1 h (1/1000, Invitrogen), counterstained with DAPI (1 μg/mL, Vector Laboratories), rinsed and mounted in Fluoromount-G for confocal microscopy or Vectashield (Vector Laboratories) for SIM. For confocal microscopy, z-stacks images (1024 × 1024 pixels, representing voxels of 0.0451 × 0.0451 × 0.198 μm) were taken using a Leica TCS SP8 AOBS confocal microscope with a 63x/NA = 1.40 oil immersion objective and × 4 zoom at ICMQuant facility. For fibroblasts, we analyzed between 26 and 32 cells in each individual, in a total of 3 euploid individuals and 3 individuals with DS.

ECs were plated in 1% porcine skin gelatin-coated Ibidi µ-slide eight-well chambers and oligofected with pEGFP tensin1. The day after, ECs were treated with FAK inhibitor PF-562271 (selleckchem.com) or DMSO for 30 min at 37°C and then analyzed by using a Leica TCS SP8 AOBS confocal microscope equipped with a HC PL APO CS2 63×/1.40 oil objective, a 37°C humidified and 5% CO₂ containing chamber, and PMTs detectors. A high-intensity 488 nm Argon laser line was used to bleach the selected ROI. Two pre-bleaches, 2 bleach, and 40 post-bleach frames were acquired at a 1.4-s interval.

The autofluorescent lysosomotropic compound MDC was recently introduced as a specific autophagolysosome marker to analyze the autophagic process [41 (link)]. After 4 passages in the absence or presence of SFN (5, 10 and 15 μM), S, R and T cells (5 × 10⁵) were resuspended in 200 µL serum-free medium containing 500 nM TQR (SelleckChem, Houston, TX, USA), and the cells were incubated at 37 °C for 45 min in the dark. After incubation, 50 µM MDC was added to cells with subsequent incubation for 1 h, at 37 °C, in the dark. The cells were washed with PBS, and the pellet was resuspended in 100 µL of PBS containing WGA conjugated with Texas Red (Invitrogen, Thermo Fisher Scientific, Eugene, OR, USA) at a final concentration of 1 µM for cell surface labeling. MDC was visualized by excitation with a 5% UV diode at 405 nm, and emitted fluorescence in the range of 515–574 nm was registered. WGA conjugated with Texas Red was visualized by excitation with a 7% white laser at 589 nm, and emitted fluorescence in the range 599–661 nm was registered. Imaging was performed on a Leica TCS SP8 AOBS confocal microscope (Leica Microsystems, Wetzlar, Germany) with an objective HC PL APO CS2 63 x/1.40 OIL.

The autofluorescent lysosomotropic compound MDC was recently introduced as a specific autophagolysosome marker to analyze the autophagic process [59 (link)]. S, R and T cells (10⁵) treated or untreated with either SFN (5, 10 and 20 μM) or AITC (10, 20 and 30 μM) were resuspended in 200 μL of PBS containing 50 μM MDC and 500 nM TQR (SelleckChem, Houston, TX, USA), and cells were incubated at 37 °C for 45 min in the dark. After incubation, the cells were washed two times with PBS. To label cell surface wheat germ agglutinin (WGA) conjugated with Texas Red (Invitrogen, ThermoFisher Scientific, Eugene, OR, USA) at a final concentration of 10 μM was added to 200 µL of PBS. MDC was visualized with a 5% UV diode at 405 nm and emission spectra in the range 515 nm–574 nm, whether (WGA) conjugated with Texas Red was stimulated with a 7% white laser at 58.9 nm and emission spectra in the range 599 nm–661 nm. Imaging was performed on a Leica TCS SP8 AOBS confocal microscope (Leica Microsystems, Germany) with an objective HC PL APO CS2 63 × /1.40 OIL.

After culturing, the cells were washed and resuspended in PBS, and then, the cells were transferred to poly-L-lysine slides (Menzel Glaser, (Thermo Fisher Scientific, Bremen, Germany). Slide-bound cells were washed twice in PBS and then fixed with 1% paraformaldehyde in PBS for 10 min. After fixation, the cells were permeabilized with 0.1% Triton-X 100 in PBS, washed in PBS, and blocked with 1% BSA in PBS for 1 h at 4 °C. The cells were then incubated with specific antibodies against both HSP90α and HSP90β (described in Section 4.6) for 1 h at 4 °C in PBS containing 1% BSA. The cells were washed twice in PBS containing 1% BSA, and donkey anti-rabbit antibody linked with Alexa Fluor 488 (Life Technologies Corporation, Wilsonville, OR, USA) in PBS containing 1% BSA was applied to the cells and incubated for 1 h at 4 °C. The samples were washed twice in PBS containing 1% BSA, and the cells were then additionally labeled with 10 mg/L of 4′-6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich, St. Louis, MO, USA) in PBS for nuclei visualization. Finally, coverslips were mounted on slides with mounting medium (80% glycerol), and the samples were observed using a Leica TCS SP8 AOBS confocal microscope (Leica Microsystems, Wetzlar, Germany).

After treatment, cells on glass slides were washed with phosphate-buffered saline (PBS; Oxoid, Lenexa, KS, USA) to remove extracellular ASC1R, and a fresh medium without Phenol Red was added. Cell membranes were stained with FM 4-64FX membrane stain (Invitrogen, Waltham, MA, USA) and visualized at 744 nm. Intracellular ASC1R was visualized at 680 nm using a Leica TCS SP8 AOBS confocal microscope (Leica Microsystems, Wetzlar, Germany).