Mitochondria were isolated from cells using a Mitochondrial extraction kit (cat. no. SM0020; Beijing Solarbio Science & Technology Co., Ltd.), according to the manufacturer's protocols. Tumescent fluid (containing 120 mmol/l KCl, 20 mmol/l MOPS, 10 mmol/l Tris-HCl and 5 mmol/l KH2PO4; pH 7.4; Beyotime Institute of Biotechnology) was added to the mitochondria and the mixture was diluted to the point where the protein concentration reached 0.25 g/l at 25°C. Subsequently, 200 µmol/l CaCl2 was added to the mitochondria and the absorbance at 520 nm was observed for 15 min. The alterations in absorbance suggested the degree of mitochondrial swelling, which indicated the opening of mPTP.
Kh2po4
Manufactured by Beyotime
Sourced in China
KH2PO4 is a chemical compound commonly used as a laboratory reagent. It is the potassium salt of dihydrogen phosphate and has a chemical formula of KH2PO4.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using kh2po4
1
Mitochondrial Swelling and mPTP Opening
2
Osteogenic Differentiation of Cells
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When the cells reached to 90% confluence, the conventional growth medium was replaced with osteogenic differentiation medium, which was prepared by 15 mL 15% foetal bovine serum [FBS (Gibco, USA)], 10 μL 10−8 mol/L dexamethasone sodium phosphate (Beyotime, China), 1 mL 1.8 mmol/L KH2PO4 (Beyotime, China), 1 mL 100 U/mL penicillin (Gibco), 1 mL 100 U/mL streptomycin (Gibco), 1 mL 0.1 mmol/L L‐ascorbic acid phosphate (Beyotime, China), 1 mL 2 mmol/L Glutamine (Beyotime, China) and Alpha MEM (Hyclone, USA) to up 100 mL. The medium was renewed every 3 days till Day 7 or Day 14. Mineral deposit was then detected by ARS Staining (Cyagen, USA). According to the manufacturers’ instructions, the cells were fixed with 2 mL of 4% neutral formaldehyde solution for 30 minutes. After removing formaldehyde and washing the plate with PBS, ARS solution was added for 5 minutes. The staining cells were observed by a light microscope (OLYMPUS, Japan).
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