Eclipse te 2000 e inverted

The shear stress was initiated by a step change from zero to 15 dyn/cm² using a parallel-plate flow chamber as described previously [7] , [33] (link), [34] (link), and was applied on RFPEC or BAEC monolayers. Briefly, the shear stress reached its steady state value rapidly after we turned on the pump which maintained the hydraulic pressure in the flow system. The circulating medium for shearing flow was DMEM (for RFPEC) or MEM (for BAECs) with 5% FBS and 0.5% BSA unless indicated otherwise. The flow system was kept at 37°C in a humidified 5%/95% CO₂/air incubator. The cell morphology was visualized using a Nikon Eclipse TE2000-E inverted microscope (Nikon) with a digital camera (Photometrics cascade 650, Roper Scientific).

HAECs were cultured on the functionalized pSi substrates for 48 h. After cell culture experiments, culture media were removed and cells were washed two times with PBS at 37°C. The cells were fixed with a 4% (w/v) solution of paraformaldehyde in PBS for 30 min at room temperature. After washing two times more with PBS, the substrates were immersed in 0.2% Triton-X 100 in PBS for 10 min at room temperature to permeabilize the cell membrane. After rinsing with PBS two times, the actin filaments and nuclei were stained in the dark at room temperature. Actin-stain 670 phalloidin (tebu-bio, Le Perray-en-Yvelines, France) was used to stain the actin filaments (200 nM, 30 min), while NucGreen Dead 488 (Life Technologies, Carlsbad, CA, USA) was used to stain the nuclei (two drops/mL, 10 min). Each sample was washed three times with PBS, and after mounting on microscope slides using anti-fade mounting media, the samples were incubated overnight in the dark at room temperature. Stained cells were kept at 4°C in the dark until microscope observations. The fluorescence images were acquired using a Nikon Eclipse TE2000-E inverted microscope (Nikon Instruments, Amsterdam, Netherlands), equipped with a C1 laser confocal system (EZ-C1 software, Nikon). Argon 488- and 633-nm lasers were used as excitation sources for NucGreen and phalloidin, respectively.

A sample of mid-log phase culture of mg491-F157A-F158A strain was diluted 200x in SP-4 and grown overnight on 8-well μ-slides ibiTreat (IBIDI). Just before visualizing cells, culture medium was replaced with fresh pre-warmed SP-4 containing Hoechst 33342 0.01 mg/ml. Cells were observed by phase contrast and epifluorescence in a Nikon Eclipse TE 2000-E inverted microscope. Phase contrast and DAPI (excitation 387/11 nm, emission 447/60 nm) epifluorescence pictures were captured with a Digital Sight DS-SMC Nikon camera controlled by NIS-Elements BR software.