Diaphot phase contrast 2 el wd 0.3 fluorescence microscope

Ex vivo mouse aortic ring assay can be utilized to study angiogenesis and the effectiveness of drugs or compounds in affecting angiogenesis.¹¹ BALB/c mice of age of 7 weeks were sacrificed and the dorsal aorta was harvested, rinsed with ice-cold PBS a few times, and cut into approximately 1.5-mm pieces. Then, each piece was inserted in a 96-well plate that had been precoated with matrigel. Another layer of matrigel was sandwiched on top afterwards and it was allowed to solidify before EGM media with camel urine at a concentration of 4 mg/mL was added to the wells. The plate was incubated in a 90% humidified incubator at 37°C with 5% CO₂ for 7 days before it was observed under Diaphot Phase Contrast-2 EL WD 0.3 fluorescence microscope (Nikon). This assay was carried out according to the protocol outlined by Baker et al.¹¹

This assay was carried out according to the protocol outlined by Liang et al.^{10 (link)} 4T1 cells were seeded at concentration of 2.4 × 10⁵ cells/mL in 6-well plates for 24 hours and incubated in a 90% humidified incubator at 37°C with 5% CO₂. After 24 hours, a sterile yellow tip was used to scratch a straight line in the middle of the wells and the media was discarded before fresh media with 3 different concentrations of camel urine—2 mg/mL, 1 mg/mL, and 0.5 mg/mL—were introduced into the wells. Images were captured using Diaphot Phase Contrast-2 EL WD 0.3 fluorescence microscope at 0, 20th, and 24th hour to determine the rate of migration (Nikon, Tokyo, Japan). The rate of migration was determined using the following formula:
$\frac{\mathrm{Area\; of\; wound\; at}0H-\mathrm{Area\; of\; wound\; at}\left(n\right)H}{\mathrm{Area\; of\; wound\; at}0H}\times 100$

In this assay, the ability of camel urine to inhibit the mortality of 4T1 cells was investigated by migration and invasion assays using culture insert (Becton Dickinson, Franklin Lakes, NJ). In summary, 4T1 cells were seeded at a concentration of 5 × 10⁵ cells/mL inside cell culture insert and treated with 3 different concentrations determined from the MTT cytotoxic assay, which were 2 mg/mL, 1 mg/mL, and 0.5 mg/mL. Three independent, biological replicates were carried out. One day before seeding and treatment, media with FBS serum was aspirated from the culture flask of 4T1, and it was replaced with media without serum. The cell culture insert was placed in each well of a 6-well plate. For invasion assay, 100 µL of matrigel (Becton-Dickinson) was added and allowed to set for an hour. Then, 4T1 cells were seeded inside the cell culture insert and treatment was added immediately before the plate was incubated in a 90% humidified incubator at 37°C with 5% CO₂ for 24 hours. Then, the insert was removed from the well and fixed with 100% methanol for 2 hours before it was stained with crystal violet for 1 hour. Stained cells were observed under Diaphot Phase Contrast-2 EL WD 0.3 fluorescence microscope (Nikon). Migrated or invaded cells were counted in each experiment in 4 different pictures taken for each replicate.