Axio observer d1 inverted microscope

Conditioned media collected from CV-MSCs with HP or non-HP after ischemic stimulus were centrifuged at 10000 rpm for 10 min to remove floating cells and debris. For survival experiments, human endothelial colony forming cells (HECFCs) were seeded in 96-well plates (20000 cells/cm²) with three independent samples in duplicates and cultured in the conditioned media for 5 days. The media were changed every other day. The viable cell number was determined at different time points using MTS assay. For migration experiments, HECFCs were seeded in Culture-Insert 2 Well in μ-Dish (Cat. No.: 81176, ibidi) with three independent samples in duplicates and cultured in the conditioned media for 12 h. Images were taken using a Carl Zeiss Axio Observer D1 inverted microscope. The cell-covered area was quantified using ImageJ software. For tube formation experiments, HECFCs were seeded in 96-well plates precoated with 50 μL Matrigel (Cat. No.: 354234, Corning) according to the manufacturer's instructions at a density of 10⁴ cells/well with three independent samples in duplicates and cultured in the conditioned media. After 12 h, cells were observed using the Carl Zeiss Axio Observer D1 inverted microscope. The total segment tube length was quantified using ImageJ software.

Cells grown on coverslips were fixed with 3% paraformaldehyde and stained with primary and fluorescently labeled secondary antibodies. The following antibodies were used: mouse anti-α-smooth muscle actin (α-SMA) (clone 1A4, Sigma, Darmstadt, Germany), mouse anti-synaptopodin (clone D-9, Santa Cruz, Santa Cruz, CA, USA), mouse anti-cytokeratin 18 (CK18) (clone RGE-53, Merck Millipore, Darmstadt, Germany), rabbit anti-fibronectin (Sigma), mouse anti-integrin α_vβ₃ (clone LM609, Millipore), mouse anti-CD31 (Dako, Glostrup, Denmark), and mouse anti-N protein PUUV (A1C5, Progen, Heidelberg, Germany). Cell nuclei were stained by Hoechst 33342 (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). Images were taken using an Axiocam 506 mono camera attached to an Axio Observer.D1 inverted microscope (Carl Zeiss, Oberkochen, Germany). For Western blot analysis, the following primary antibodies were used: rabbit anti-PUUV N protein and mouse anti-α-tubulin (Sigma). Loading was verified by the detection of tubulin on the same membrane. Detection was performed by using near infrared fluorescent dye (IRDye)-conjugated secondary antibodies and an Odyssey CLx infrared imaging system (Li-Cor, Lincoln, NE, USA).

GJ permeability was evaluated at room temperature (RT) using the scrape loading/dye transfer (SL/DT) technique [93 (link),94 (link)]. Briefly, MCs cultures were washed for 10 min in HEPES-buffered salt solution containing the following (in mM): 140 NaCl, 5.5 KCl, 1.8 CaCl₂, 1 MgCl₂, 5 glucose, 10 HEPES, pH 7.4, followed by washing in a Ca²⁺-free HEPES solution for 1 min. Then, a razor blade cut was made in the monolayer in a HEPES-buffered salt solution with normal Ca²⁺ concentration containing the fluorescent dye Lucifer yellow (LY). After 1 min, LY (100 μM) was washed out several times with HEPES buffered salt solution. At 8 min after scraping, fluorescent images were captured using a Zeiss Axio Observer D.1 Inverted Microscope with a Solid-State Colibri 7 LED illuminator and with a 10× objective. Changes were monitored using an AxioCam MRm monochrome digital camera R3.0 (Carl Zeiss AG, Zeiss, Oberkochen, Germany), and Software ZEN Pro (Zen 2.3 [blue edition], Carl Zeiss AG, Oberkochen, Germany) for image acquisition and analysis. For each trial, data were quantified by measuring fluorescence areas in three representative fields. Quantification of changes in GJ communication induced by different treatments was performed by measuring the fluorescence area, expressed as AU [93 (link),94 (link)].