Earle s balanced salt solution

Caudae epididymis from each mouse was placed in 2 mL Earle's balanced salt solution (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 0.1% bovine serum albumin (Sigma-Aldrich). The epididymis was gently teased with a bent needle to release spermatozoa under observation through a stereomicroscope (Olympus). Sperm density was assessed with a haemocytometer and was presented by spermatozoa count per epididymis [36 (link), 37 (link)].

C2C12 myoblasts were cultured in a growth medium (DMEM; (Wako) supplemented with 10% FBS and 1% penicillin-streptomycin) at 37 °C with 5% CO₂ as previously described [52 (link)]. Myogenic differentiation was induced by changing the growth medium to a differentiation medium (DMEM supplemented with 2% horse serum and 1% penicillin-streptomycin) at 37 °C with 5% CO₂. For Western blotting, myotubes were deprived of serum for 8 h, and then cultured for an additional 2 h in Earle’s Balanced Salt Solution (Sigma-Aldrich) to deprive both serum and amino acids. C2C12 myotubes were treated with the indicated reagents for 30 min.

Lung tissues were collected at necropsy (56 dpi) and stored at −80°C until they were processed for PCR testing. As described elsewhere (18 (link)), lung (3 by 3 cm) containing both normal and affected tissue was minced using sterile scissors and then placed in a 50-ml conical tube with 30 ml of Earle’s balanced salt solution (Sigma-Aldrich, St. Louis, MO, USA) at a concentration of 10% (wt/vol). The sample was homogenized (2 min at 1,000 rpm; Geno/Grinder; Spex SamplePrep, Metuchen, NJ, USA) and then centrifuged (10 min at 4,200 × g).
M. hyopneumoniae DNA detection for both tracheal and lung homogenates was based on commercial kits (MagMax-96 Pathogen RNA/DNA kit, PCR VetMax-Plus qPCR master mix, VetMax Mycoplasma hyopneumoniae reagents; Applied Biosystems) performed as directed by the manufacturer. DNA was extracted on the Kingfisher Flex system and amplified on Applied Biosystems 7500 real-time PCR. Each plate included a known M. hyopneumoniae-positive sample (VetMax-Plus qPCR master mix kit includes Xeno DNA Control, Applied Biosystems) and a negative control sample (RNA-free water). A test result was considered valid when the internal positive C_T value was ≤36. A sample was considered M. hyopneumoniae positive when C_T values were ≤37.

HeLa and 293T cell lines (ATCC) were cultured in DMEM containing 10% FBS and antibiotics (100 μg/mL streptomycin and 100 units penicillin). The FLCN-null UOK-257 and FLCN-restored UOK-257-2 human clear-cell renal cancer cell lines were obtained from Dr. Laura S. Schmidt (National Cancer Institute, Bethesda, MD, USA) and cultured as described previously [11 (link), 54 (link)]. HeLa and 293T cells were transfected with Lipofectamine (Invitrogen, Carlsbad, CA, USA) in Opti-MEM (Invitrogen) according to the manufacturer's instructions. For the half-life experiment, transfected cells were split into 60-mm tissue culture dishes at 24 h after transfection, and treated with 100 μg/mL cycloheximide (Sigma-Aldrich, St Louis, MO, USA) the next day. At indicated time points, cells were harvested for western blot analysis. Packaging of lentiviruses, retroviruses, and subsequent infections of various cell lines were performed as described previously [55 (link)]. Where indicated, HeLa, UOK-257, or UOK-257-2 cells were starved in Earle's Balanced Salt solution (Sigma-Aldrich) for 3 h and stimulated with 10% FBS, glucose-free or glutamine-free DMEM (Gibco) for a subsequent western blot or immunofluorescence analysis.

Subjects were instructed to abstain from ejaculation for 3 to 5 days prior to sample collection. Semen samples were obtained via masturbation, collected in disposable sterile containers, and kept at room temperature until liquefaction occurred. Semen analysis was completed using a computer-assisted semen analyzer. Sperm were isolated from semen samples by using Percoll gradient centrifugation [37 (link)]. Sperm samples were incubated at 37°C for one hour in Earle’s balanced salt solution (SigmaAldrich, St. Louis, MO) [36 , 38 (link)] before utilization.

As described (Li et al., 2020), the rats were euthanatized and decapitated, L4–L6 DRGs of control limbs and occluded limbs were removed and dissected, immediately transferred into ice‐cold Hank's balanced salt solution. After freeing from the connective tissues, the ganglia were enzymatically digested and dissociated in Earle's balanced salt solution (Sigma‐Aldrich) containing collagenase Type D (0.6 mg/ml; Roche), trypsin (0.30 mg/ml; Worthington), and DNase (0.1 mg/ml; Alfa Aesar), followed by shaking for 40 min at 34°C. The dissociated neurons were seeded on 10% poly‐L‐lysine–coated coverslips (Dia^# 8mm) in a 35‐mm culture dish containing 2 ml of DMEM medium (Thermo) supplemented with 10% FBS, 1% glutamine, and 1% penicillin–streptomycin. Then the neurons were cultured at 37°C with 5% CO2, 95% air in a cell culture incubator (VWR). Then, IL‐6, homo IL‐6/IL‐6Rα fusion protein (H. IL‐6/6Rα) and SC144 were added to the culture solution in this experiment, respectively, as detailed in the results. The dosages of those agents were selected according to our pilot experiments and previous reports (Fang et al., 2015; Liu, Chen, et al., 2019; Xia et al., 2015). Then, the patch recording was completed within 6 to 48 h after DRG neurons were dissociated.