Multimode reader

A caspase-3 activity assay kit (cat. no. BB-4106; BestBio Company) was used to detect the activity of caspase-3 in different groups, according to the manufacturer's protocol. A multimode reader (Thermo Fisher Scientific, Inc.) was used to measure the absorbance at 405 nm of different samples.

The epididymitis model was examined by real-time PCR and ELISA. Frozen epididymal samples from rats in the model and sham-operated groups were homogenized in Trizol (Invitrogen, Carlsbad, CA, USA) in a tissue homogenizer (Tiangen Biotech Co., Ltd., Beijing, China). Total RNA was extracted and reverse-transcribed with a PrimeScript RT Master Mix (Takara Biotech Co., Ltd., Dalian, China) in a total volume of 20 μl. cDNA samples were amplified with SYBR Green PCR Master Mix (Thermo Fisher Scientific, Inc., Carlsbad, CA, USA) in a 7500 real-time PCR system (Applied Biosystems, Foster City, CA, USA) under the following cycle conditions: 50°C for 2 min, 95°C for 2 min, and 40 cycles of 95°C (15 s) and 60°C (60 s). Primers used for the PCR experiments are displayed in Supplementary Table 1. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the reference gene to calculate the relative expression of each target gene.
The protein concentrations of IL1β, TNF-α, and IL6 in epididymitis tissue samples were analyzed via ELISA kits from Thermo Fisher Scientific according to the manufacturer's instructions. A multimode reader (Thermo Fisher Scientific) was used to assess the absorbance at 450 nm. Each measurement was performed in triplicate.

The level of cellular ROS induced by the EA extract of C. gloeosporioides in human breast cancer cells MDA-MB-231
and MCF-7 was evaluated using the 2′, 7′-dichlorofluorescein
diacetate (H2DCFDA) dye-based assay as described previously.^{30 (link)} Briefly, human breast cancer cells, MDA-MB-231
and MCF-7, at a density of 2 × 10⁵ cells were treated
with the EA extract of C. gloeosporioides with a respective IC₅₀ value for 24 h. The treated cells
were harvested and homogenized in 1X PBS, followed by centrifugation
at 10,000g at 4 °C. The supernatant containing
cellular ROS was collected and quantified using a NanoDrop ONE (Thermo
Scientific, USA). Further, an equal quantity of protein was incubated
with 2 mM 2′,7′-dichlorofluorescein diacetate (H2DCFDA)
for 30 min at 37 °C. The available ROS converted the non-fluorescent
H2DCFDA to highly fluorescent 2′,7′-dichlorofluorescein
(DCF), which was recorded at an excitation wavelength of 488 nm and
an emission wavelength of 525 nm using a multimode reader (Thermo
Scientific, USA). The level of cellular ROS in each sample was determined
by calculating the fluorescence intensity value per milligram of protein.
The representative result was from three independent experiments.