Unicel dxl 800

The blood samples of mice were collected after 6 hours of fasting. Then left for 30 minutes to centrifuge at 3000 r/min for 10 minutes, and the serum was collected for analysis. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), total cholesterol (TCH), triglyceride (TG), high-density lipoprotein (HDL) and low-density lipoprotein (LDL) levels in mice were measured using UniCel Dxl800 (Beckman Coulter).

Testosterone circulates in several forms: unbound or “free”, loosely bound to albumin, or tightly bound to SHGB. Bioavailable testosterone refers to the first two categories, which are available for biological processes and hence more relevant to purported causal effects. The primary exposure in this analysis is bioavailable testosterone, but in additional analyses, we examined relationships with total and free testosterone. In the UK Biobank, testosterone and SHBG were measured by one-step competitive analysis on a Beckman Coulter Unicel Dxl 800 in nM, and albumin was measured by bromocresol green (BCG) analysis on a Beckman Coulter AU5800 in grams per liter. There were 23 participants with the minimum detectable limit of 0.35 nM testosterone; we coded these participants as having 0.35 nM testosterone. All participants had over the minimum detectable levels of albumin and SHBG.
We estimated free testosterone using the method detailed in Ho et al. (50 (link)), based on the work of Vermeulen et al. (51 (link)) $\text{FT}=\frac{T-0.5217A-S-1+\sqrt{{(0.5217A+1+S-T)}^2+4T\times (0.5217A+1)}}{2\times (0.5217A+1)}$ where FT is free testosterone, T is total testosterone (nanomoles per liter), S is SHBG (nanomoles per liter), and A is albumin (grams per liter). Similarly, we estimated bioavailable testosterone as $\text{BAT}=\text{FT}+0.5217A\times \text{FT}$ where BAT is bioavailable testosterone.

The clinical parameters, including age, BMI (= weight[kg]/height[m]²) and the number of days in the menstrual cycle of all individuals were evaluated by a research assistant. Parameters (including progesterone, estradiol, LH, FSH, testosterone, prolactin, androstenedione, LDL-C, TC, TG and HDL-C) were analyzed by electrochemiluminescence immunoassay (Roche Diagnostics). The levels of TC ≥ 5.2 mmol/L, TG ≥ 1.7 mmol/L, HDL-C ≤ 1.0 mmol/L, and LDL-C ≥ 3.4 mmol/L, was defined as dyslipidemia according to the Chinese guidelines for lipid management (2023) [61 (link)].DHEA-S and SHBG were measured by chemiluminescence immunoassay (Beckman Coulter Unicel Dxl 800) Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were analyzed by a biochemical analyzer (Liaison XL, Diasorin, Saluggia, Italy).

Measurement of serum 25(OH)D was performed by chemiluminescence immunoassay UNICEL Dxl 800, using a fully automatic analyzer Beckman Coulter. Interpretations of 25(OH)D concentrations (according to the reagent kit protocol) were as follows: deficient if <20 ng/mL, insufficient if between 20 and 30 ng/mL, and sufficient if ≥ with 30 ng/mL Similar levels are recommended by the American Endocrine Society [12 (link)].

The cellular proteins were extracted form skeletal muscle cells and lysed by RIPA lysate (R0010, Solarbio, Peking, China). Equal amounts of protein were electrophoresed on sodium lauryl sulfate polyacrylamide gel (SDS-PAGE) with a loading of 20 μL per well. The protein was then transferred to an activated PVDF membrane (FFP26, Beyotime, Shanghai, China) and blocked with 5% skim milk powder (P0216, Beyotime, Shanghai, China) for 2 h. Appropriately diluted primary antibodies against Beclin-1 (Abcam, Cat# ab223372), p62 (Abcam, Cat# ab101266), p-mTOR (Abcam, Cat# ab109268), mTOR (Abcam, Cat# ab2732), p-AKT^Ser473 (Abcam, Cat# ab18206), p-IRS1^Ser616 (CST, Cat# 2386S), IRS (CST, Cat# 2382S), AKT (CST, Cat# 2938S), LC3 (Affinity, Cat# AF5402) and β-actin (Abcam, Cat# ab8227) were added and incubated at 4 °C overnight. After washing the membrane, the corresponding secondary antibody (Cat# A0208 and A0216, Beyotime, Shanghai, China) was added for incubation. Chemiluminescence detection was then carried out using an enhanced chemiluminescence reagent (P0018AS, Beyotime, Shanghai, China). After development and fixing treatment, the film was photographed by a gel imaging analysis system (UniCel Dxl800, Beckman Coulter, Pasadena, CA, USA).