Dm14000b

Spheroids were generated by gravity as described [24 (link)]. Briefly, spheroids were embedded into a collagen gel, and then rapidly transferred into a 24-well plate and allowed to polymerize at 37 °C incubators for 30 min, M199 medium with or without VEGF and Andro was applied on top of the gel. After cultured for 24 h, spheroid sprouts were evaluated by measuring the cumulative length of all capillary like sprouts using a Leica DM14000B microscope. At least 5 randomly selected spheroids per experimental group and experiment were analyzed. And sprout length was measured with Image J software.

Following density gradient separation, peripheral blood mononuclear cells from a healthy donor were harvested and monocytes were isolated using human CD14 MicroBeads UltraPure (130-118-906; Miltenyi Biotec, Germany), according to the manufacturer’s instructions.^{25 (link)} CellTracker Red CMTPX (C34552; Invitrogen) at a concentration of 2 µM was used to fluorescently label the monocytes, according to the manufacturer’s instructions. ECs were cultured under flow using the orbital shaker model as described above and stimulated with TNF (10 ng/mL) for the last 4 hours of flow. The media was removed and replaced with media containing fluorescently labeled monocytes (1×10⁶ per well) and the cells were incubated for 2 hours under static conditions. The wells were washed gently with PBS to remove nonadherent monocytes and fixed in paraformaldehyde (4% w/v). DAPI (Sigma) was used to label the nuclei. Fluorescent images were taken using the ×20 objective of a wide-field microscope (DM14000B; Leica) to detect adherent monocytes. An average of 6 images were analyzed per well and used to calculate the average number of adherent monocytes per EC per sample.

HCAEC were cultured under OSS for 72 h as described above. Unlabelled NMVs (2 × 10³ µL⁻¹) were perfused over the cells under OSS for 2 or 4 h. The media was removed and replaced with media containing fluorescently labelled monocytes (1 × 10³ µL⁻¹) and this was perfused over the HCAEC for 2 h under OSS. Slides were washed gently to remove non-adherent monocytes and fixed in paraformaldehyde (4% w/v). Phase-contrast and fluorescent images were taken using the ×10 objective of a wide-field microscope (Leica, DM14000B) to detect adherent monocytes. An average of 15 images were analysed per slide and used to calculate the number of adherent monocytes per field of view per sample.

HK-2 cells were seeded in 6-well plates and cultured overnight. Fresh medium containing IMP (2 mM) in the absence or presence of JBP485 (500 μM) or cilastatin (500 μM) was then added, and the cells were incubated for an additional 24 h. Then, the cells were stained with JC-1 (2 μM, Beyotime Institute of Biotechnology, Shanghai, China) and observed by a fluorescence microscope (Leica DM 14000B, Germany).

The cells were seeded into six-well plates and incubated in the medium with 2.5% FBS until grown to full confluency, then scraped by a sterile 200 μL pipette tip. The medium was replaced with PBS, and the wound gap was photographed with an inverted microscope with a digital camera (Leica DM 14000B, Wetzlar, Germany) at 0 h and 24 h.

HCAEC were cultured under static conditions, HSS or OSS for 72 h as described above. Fresh complete growth medium containing fluorescently labelled NMVs (1 × 10³ µL⁻¹) was added to cells under static conditions or perfused over Ibidi µ-slides for 2 h at 37 °C and 5% CO₂ under the same shear conditions used for pre-conditioning the cells. Following incubation, the medium was removed and cells gently washed three times with PBS to remove residual NMVs. Phase-contrast and fluorescent images were taken using the ×20 lens of a wide-field microscope (Leica, DM14000B) and the mean number of fluorescent NMVs in six fields of view per sample was calculated.
To assess NMV adhesion to monocytes, fluorescently labelled NMVs (1 × 10³ µL⁻¹) were incubated with 2 × 10⁵ monocytes for 2 h at 37 °C. Unbound NMVs were removed by centrifugation (400 × g for 6 min) and the cells washed. NMV adhesion was analysed using an LSRII flow cytometer and data analysed for changes in mean fluorescence intensity using FACSDiva acquisition software.