Jca bm6050

On E18.5, amniotic fluid was aspirated from gestational sacs using a tuberculin syringe with a 20-gauge needle before opening the fetal membranes. Amniotic fluid was centrifuged for 10 min at 2,700 g and 20 min at 15,800 g to remove cellular debris. When the recovered fluid was more than 50 μl, the amniotic fluid was just used to determine osmolality and the concentrations of electrolytes, otherwise the amniotic fluid from the same genotype littermates was mixed and used in experiments. Fifty microliters of amniotic fluid was diluted to 200 μl with MilliQ water. Amniotic fluid osmolality was measured by freezing point depression (OSMO Station OM-6050, ARKRAY Inc.). Na⁺, K⁺, and Cl⁻ concentrations were determined with JCA-BM6050 (JEOL Ltd.).

For the measurement of renal ATP content, a 30 mg kidney sample was homogenized in 600 μL of ultrapure water and centrifuged (10,000× g at 4 °C for 10 min). Then, the ATP concentration in the supernatant was measured using a Tissue ATP assay kit (Toyo B-Net, Tokyo, Japan) according to the manufacturer’s instructions. Blood tests were performed using a JCA-BM6070 clinical analyzer (JEOL, Tokyo, Japan). BUN, serum Cr, FFA, TG, total protein, albumin, and ALT levels were measured by standard methods. Serum HDL and low-density lipoprotein levels were evaluated by the direct homogeneous assay method. Urine tests were performed with a JCA-BM6050 clinical analyzer (JEOL, Tokyo, Japan). Urine total protein levels were determined by the pyrogallol Red assay method. Urine NAG and Cr concentrations were measured by the enzyme assay method. Renal FFA was extracted using the hexane/isopropanol method as previously described [42 (link)]. Briefly, a 20 mg kidney sample was homogenized in 100 μL of ice-cold phosphate-buffered saline. Lipids were extracted from the homogenate with 900 μL of n-hexane:isopropanol (3:2, v/v) and dried using an evaporator. The dried lipids were solved with a 0.1% solution of Triton X-100 in ultrapure water (100 μL), and then FFA content was measured by means of NEFA C-test kits (Wako Pure Chemical, Osaka, Japan).

Total LDH activity (IU/l) was measured by an autoanalyzer JCA-BM6050 (JEOL, Tokyo, Japan) using enzymatic method L-Type Wako LD IF or L-Type Wako J (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). The percentages of LDH isozymes were measured using a Hydrasys 2 Scan (Sebia, Paris, France) with a HYDRAGEL 7 ISO-LDH (Sebia).
Measurements of total LDH activity and LDH isozymes were performed by a clinical laboratory testing company, Fujifilm Vet Systems (Tokyo, Japan).