Ix73p1f

Transfected HUVECs were detached from the cell culture plates using trypsin–EDTA solution, and subsequently suspended in ECM medium enriched with 10% FBS. Following this, a total of 8 × 10³ cells were cautiously mixed with 4 ml of cell culture medium and 1 ml of methocel stock solution, which contained 12 mg of methylcellulose (Aladdin, C104984-250 g). This resulted in the formation of spheroids, which were obtained by placing 25 μl drops of the cell solution onto the lid of a cell culture dish (100 × 20 mm), and then incubating the dish upside-down in a humidified cell culture incubator for 24 h. The hanging drops were then carefully washed off using phosphate-buffered saline. Subsequently, the HUVECs were resuspended in 2 ml of methocel solution containing FBS, and 1 ml each of PBS and rat collagen I (R&D system, Shanghai, China) was added gently. The resulting spheroid-collagen solution was added to 24-well plates (1 ml per well) for 24 h. The sprouts were captured using an inverted microscope (Olympus Corp, IX73P1F) and the number and average length of each sprout per cytosphere were determined using the Sprout Morphology plugin in Fiji (ImageJ).

The identification of EPC phenotypes was done by using fluorescence microscopy and flow cytometry. For the detection by fluorescence microscopy, both double-positive staining and immunofluorescent staining were performed. After 7 days of culture, EPCs were incubated with Dil-conjugated acetylated low-density lipoprotein (Dil-ac-LDL, L3484, Invitrogen) and Ulex Europaeus Agglutinin I (UEA-I, L9006, Sigma-Aldrich). Double-positive staining for Dil-ac-LDL and UEA-l was considered to represent EPCs and counted at × 40 magnification under a fluorescence microscope (IX73P1F, Olympus). For extracellular labeling of CD133, CD34, and VEGFR2, EPCs were fixed in 4% PFA for 30 min; blocked with 5% bovine serum albumin; and labeled sequentially with the antibodies against CD133, CD34, and VEGFR2, respectively (1:200, Santa Cruz Biotechnology). They were subsequently washed three times in PBS and counterstained with diamidino-phenylindole for 5 min. Following washing, the images were captured by fluorescence microscopy. For the detection by flow cytometry, EPCs were digested with 0.25% trypsin and permeabilized with 0.1% Triton-X at 37°C for 30 min. Subsequently, these cells were incubated with antibodies against CD133, CD34, or VEGFR2 (1:50, Santa Cruz Biotechnology); detected by a CytoFLEX flow cytometer (Beckman Coulter); and analyzed with the CytExpert software.

At each time point, eTCs were stained with 4 μM calcein-AM and 2 μM ethidium homodimer-1 in HBSS. After 30 min incubation at 37 °C in a humidified atmosphere of 5% CO₂, the staining solution was replaced with HBSS. Three images per replicate were acquired using FITC and TRITC filters of an Olympus IX73P1F (Olympus Corporation, Tokyo, Japan) inverted fluorescence microscope at 10× magnification. Using ImageJ (NIH, USA) software, alive (calcein positive, green fluorescent) and dead (ethidium positive, red fluorescent) cells were counted from the corresponding fields. The average percentage of live cells to total cells was calculated.

The synchronized eggs were placed on the NGM plates with ZGE supernatant concentrations of 0, 0.8125, 1.625, 3.25 and 6.5 mg/mL, and cultured at 20 °C with 100 μM FUDR in each group. When the body length of the nemotodes was no longer growing, each group of nematodes was picked up to add to the glass slides containing 0.1% sodium azide. The nematode was straightened with the tip of the gun. The nematodes were observed by Olympus IX73P1F fluorescence microscopy and the length of each group was calculated.

BrdU was performed as previously described [63 (link)]. In each experiment, ten animals were used in each group. Animals were treated with 1× Montjuic salts with 0.0625% N-acetylcysteine for 30–60 s three times and washed in 1× Montjuic salts for 1 min. Then, the animals were incubated in 1× Montjuic salts with 5 mg/mL BrdU (Sigma, Shanghai, China) for 1–2 h in the dark at 21 °C. After maintenance in 1× Montjuic salts at room temperature for 6–10 h, the animals were killed in 5% NAC for 5 min and fixed in 4% paraformaldehyde for 30 min at room temperature. Thereafter, 6% hydrogen peroxide in methanol was used to bleach the animals under bright light overnight. Next, the animals were rehydrated through a methanol dilution series in PBST and were treated with 2N HCl at room temperature for 45 min. After washing three times with PBST and blocking in PBST with 0.25% BSA at room temperature for 6 h, the animals were incubated in 1:1000 rat anti-BrdU (Proteintech, Beijing, China) overnight and washed in PBST eight times over 6 h the next day. Moreover, 1:500 goat anti-rat conjugated to HRP (Sangon Biotech, Shanghai, China) was used to label rat anti-BrdU at room temperature overnight. Finally, the animals were treated with tyramide conjugated to Alexa568 (Molecular Probes) for 30 min and were observed by NIS element software (version 4.2.0, Olympus, IX73P1F, Tokyo, Japan).