Evos fl digital inverted microscope

Cells were treated with 1 µg/mL TNF-α and 250 ng/mL cycloheximide for 2 h at 37 °C. In cases where inhibitors or activators were used, they were added at the same time as TNF-α and CHX. Following treatment, cells were prepared for staining by washing off spent media and chemicals from the cells with warm PBS. Cells were spun down at 400× g for 10 min and the cell pellets were resuspended in warm RPMI 1640 + 10% FBS containing 2.5 μg/mL Hoechst 33342 (Millipore-Sigma, Burlington, MA, USA) and 100 μL/1 mL from a 1:100 dilution of JC-1 Assay Reagent (Cayman Chemical Company, Ann Arbor, MI, USA). The cells were incubated for 22 min at 37 °C and centrifuged at 400× g for 10 min. Cell pellets were resuspended in freshly warmed RPMI 1640 + 10% FBS and diluted 1:1 in a 6-well plate to be imaged on the EVOS FL Digital Inverted Microscope (ThermoFisher, Waltham, MA, USA). Cells were analyzed with the included DAPI, GFP, and RFP fluorescence filters and then presented as overlay images. Fluorescence intensity was measured using ImageJ.

All qualitative images shown were acquired on a Leica SP5 confocal microscope using Leica imaging software. For quantitative image analysis, three representative images were acquired across all 36 patients on an EVOS FL digital inverted microscope (Life Technologies) for both ACE2/cTnT and ACE2/α‐smooth muscle cell actin (SMA) co‐staining.

Primary human foreskin fibroblasts (Promocell) were cultured until confluent in 48-well plates in Q333 fibroblast growth media (PAA Laboratories, Pasching, Austria). 7500 endothelial cells were then seeded per well onto the fibroblasts monolayer in a 1:1 mixture of Q333 and ECGM and left to acclimatize for 24 h. Media was then aspirated and replaced with fresh ECGM±VEGF-A isoform (1.25 nM) as desired; media was replaced every 2-3 days for seven days. Co-cultures were then fixed in 200 µl 10% (v/v) formalin for 20 min and blocked in 5% (w/v) BSA for 30 min at room temperature. Co-cultures were then incubated with 1 µg/ml mouse anti-human PECAM-1 (CD31; Santa Cruz, USA) overnight at 4°C. Cells were washed three times with PBS before incubation with an anti-mouse Alexa Fluor 594 conjugate (Invitrogen) for 3 h at room temperature. Wells were then washed three times with PBS. Endothelial tubules were visualized by immunofluorescence microscopy using an EVOS-fl inverted digital microscope (Life Technologies, Paisley, UK). Five random fields were imaged per well. Both the number of branch points and the total tubule length was then quantified from each photographic field using the open source software AngioQuant (www.cs.tut.fi/sgn/csb/angioquant) and values averaged. For a more detailed method please see Fearnley et al., (2014b (link)).