Determination of SS31 concentration in main organs of AKI mice

1 The ICR mice subjected to renal IR were divided into (i) SS31 group, (ii) C-TK-SS31 group, and (iii) SC-TK-SS31 group.

2 At 4 hours after injection of formulations at a dose of SS31 (2 mg/kg), mice were euthanized, and main organs (kidney, heart, lung, liver, and spleen) were harvested.

3 Treatment of tissue samples：

1)     10% Tissue homogenate. The tissue blocks were rinsed in cold normal saline to remove blood, dried with filter paper, weighed and put into 5 ml centrifuge tube. The total volume of pre cooled PBS should be 9 times of the weight of tissue block. Use an ophthalmic scissors to cut up the tissue mass as soon as possible. 10% tissue homogenate (homogenizing 10 s at a time, 30 s intermittently, 3-5 times) was prepared by the internal tissue homogenizer in the ice-water bath. The prepared 10% tissue homogenate was centrifuged at 4000 rpm for 10 min. Take the supernatant.

2)     Deproteinization with CH3CN. The supernatant above was deproteinized with a 1.5 volume of acetonitrile. The mixture was centrifuged (10 min, 12000 rpm, ambient temperature), and the supernatant was evaporated under a gentle stream of nitrogen.

3)     Reconstitute. The deproteinized residue was reconstituted in 100 mM H₂O₂ to concentrate five times. Suspend for 30s and let stand for 10 min. The mixture was centrifuged (10 min, 12000 rpm, ambient temperature). Take the supernatant.

4)     Determination by HPLC. The supernatant was determined by HPLC. HPLC conditions: SS31 was quantified by HPLC with a C18 column. Acetonitrile/water with 0.1% TFA (25:75, v/v) was used as the mobile phase. The column temperature and the detection wavelength were 25°C and 220 nm with a flow rate at 1.0 ml/min and an injection volume of 20 ul.

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