Ep tube

The experimental rats from each group were fasted for 12 hours but could drink water. They were comatose by intraperitoneal injection of 1% sodium pentobarbital, and then, the ileum tissue was found, washed, and absorbed water. Two rats from each group were taken out and their intestinal mucosal tissues were scraped from partial ileum. Next, the tissues were smeared on a glass slice, which was fixed with 4°C and 4% paraformaldehyde. Then, the remaining part of the intestinal mucosa of the ileum was collected, put into a 1.5 mL microcentrifuge tube (Eppendorf (EP) tube), and stored in a refrigerator at −80°C for later use. Afterward, the rats were sacrificed, the liver was quickly separated, the left lateral lobe was taken out, and the blood stains were washed with phosphate buffer saline (PBS) for biochemical testing. Besides, the middle lobe of the liver was cut into 10 mm × 10 mm tissue and placed in 4% paraformaldehyde solution for fixation, which was used for pathological observation.

All participants adopted appropriate ovulation induction treatment to promote ovulation according to their personal conditions. In our research center, the first‐line treatment for endometriosis is the long GnRH‐α protocol. Follicular development and serum sex hormone levels were monitored regularly until the day of chorionic gonadotrophin (HCG, Lizhu, China) injection. In the process of ovulation induction, the drug dosage was adjusted according to follicular development and hormone levels.
During regular monitoring of ovulation, when at least two follicles in the bilateral ovaries were larger than 18 mm, 10,000 U HCG was injected intramuscularly to induce the final maturation of the follicles. Thirty‐four to thirty‐five hours after HCG injection, oocytes and FF were collected by puncturing mature follicles larger than 18 mm under ultrasound guidance. The oocytes were cultured in the medium, and the FF was transferred to the Eppendorf tube (EP tube). The collected FF was centrifuged at 4°C and 15,000 rpm for 10 min. The supernatant was cryopreserved at −80°C for further analysis.

Before the gas chromatography–mass spectrometry (GC‒MS) analysis, acetonitrile was added to the urine samples, and they were vortexed for approximately five minutes. NaCl was then added, and the mixture was vortexed for approximately one minute. The supernatant was pipetted into a rotary steaming flask, evaporated until nearly dry, and then diluted to a volume of 1 ml with ethyl acetate. This solution was transferred to an Eppendorf (EP) tube with 50 mg of primary secondary amine (PSA), vortexed for 30 s, and centrifuged at 8,000 r/min for four minutes. Finally, the supernatant was passed through a 0.22-μm filter, and GC‒MS analysis was performed.
Measurements of the CPF levels were performed as described elsewhere using GC‒MS analysis (Agilent GC7890A-5975). Laboratory quality control parameters for CPF levels were verified with reference to NY/T 788-2004 Pesticide Residue Test guidelines, which is the agricultural industry standard in China.

Serum samples were thawed at 4°C on ice. 100 μL of sample was taken and placed in a Eppendorf EP tube, extracted with 300 μL of methanol including 10 μL internal standard (0.5 mg/mL 2-chloro-L-phenylalanine), followed by vortex for 30 s, and then ultrasound treated for 10 min (incubated in ice water) and incubation for 1 h at −20°C to precipitate proteins and then centrifuged at 12000 rpm for 15 min at 4°C. 200 μl supernatants were transferred to LC-MS vials, 20 μL was taken from each sample and pooling as QC samples, and 200 μl QC sample were taken for the metabolomic analysis.

RT-PCR was performed to examine the gene expression of the cell-encapsulated hydrogels [39 (link)]. The samples were cultured in the DMEM/F-12 for 3 and 21 days. The samples were washed with PBS 3 times. The cell lysis of the samples was performed by homogenizing with Trizol (Takara Bio Inc., Shiga, Japan) using a glass tissue grinder (Weaton Industries, Millville, NJ, USA). The homogenized solutions were transferred to a 1.5 mL Eppendorf tube (EP tube, Eppendorf, Germany) and chloroform (Samchun chemicals, Gangnam-gu, Korea) was included. The samples were centrifuged at 12,000× g and 4 °C for 15 min. The supernatant was transferred to a 1.5 mL EP tube and isopropanol was added and stored at 4 °C for 4 h. After 4 h, the samples were centrifuged at 12,000× g and 4 °C for 15 min. The supernatant was removed and 75% of ice-cold ethanol was added and centrifuged at 7000× g and 4 °C for 5 min. The extracted mRNA was diluted with free water (UltraPure^TM Distilled Water, Life technologies, USA). The mRNA concentration was analyzed using the BioSpectrophotometer (Eppendorf, Germany) and amplification was performed with a PCR Thermal Cycler (Takara Bio Inc., Japan). The RT-PCR was carried out with an SYBR™ Green PCR Master Mix (Applied Biosystems, Middlesex, MA, USA) and StepOnePlus Real-Time PCR system (Applied Biosystems, USA). All genes were normalized by the housekeeping gene GAPDH.

Eye orbital venous blood samples were collected into eppendorf tube (EP tube) which is coated with heparin sodium from the rats on day 7 after establishing the cervicitis model. Plasma was obtained after 20 min by centrifugation at 4,500 r/min for 10 min at 4°C. All plasma samples were stored at −80°C prior to sample preparation.
A working IS solution of 2-chloro-L-phenylalanine (5.31 μg/mL) was prepared in methanol. Plasma samples (50 μL) were added to 200 μL of the working IS solution. A total of 780 μL rat plasma sample (20 μL of each rat plasma sample, one from model group died in the experimental progress) were added to 3,120 μL of the working IS solution to generate a quality control (QC) sample for validating the reproducibility of the method and UPLC-QTOF-MS/MS stability. Pretreated samples were vortexed for 3 min, and then centrifuged (15,000 r/min, 4°C) for 10 min. The supernatant was transferred into a sample bottle and stored at 4°C for MS analysis of cervicitis rat metabolomics.