Dna rna oxidative damage high sensitivity elisa kit

Ox-mtDNA for treatment was synthesized from mtDNA extracted using the Mitochondrial Extraction Kit according to the manufacture’s protocol (Active Motif, Carlsbad CA, USA) and amplified by ND1 primers (ND1 Forward: 5′-CCCTAAAACCCGCCACATCT-3′; ND1 Reverse: 5′-GAGCGATGGTGAGAGCTAAGGT-3′) with the addition of oxidized guanosine to the master mix and mtDNA amplified, as described in [13 (link)] (Supplemental Figure S1). The pyroptotic TLR4 signaling pathway was activated by incubation with LPS, ATP, and nigericin (LAN) [23 (link)]. Caspase-Glo^® 1 Inflammasome and LDH-Glo^® Cytotoxicity assays (Promega Corporation, Madison, WI, USA) were used to assess inflammasome activation following manufacturer’s protocols. Ox-mtDNA levels were quantified using the DNA/RNA Oxidative Damage (High Sensitivity) ELISA Kit (Cayman Chemical Company, Ann Arbor, MI, USA) [19 (link)].

The IMA, AGE, A1AT, and asprosin concentrations were measured using an enzyme-linked immunosorbent assay (ELISA) (Enzyme-linked Immunosorbent Assay Kit; Cloud-Clone Corp., Wuhan, China; CEA825Hu, CEB353Ge, SEB697Hu, and SEA332Hu, respectively) according to the manufacturer’s instructions. The DNA/RNA OSDP concentrations were assayed using an immunoassay kit (DNA/RNA Oxidative Damage (High Sensitivity) ELISA Kit, Cayman Chemicals, Ann Arbor, Michigan, MI, USA, 589320). This kit enabled the simultaneous detection of DNA/RNA OSDPs, such as 8-hydroxyguanosine (8-OHG), 8-hydroxy-2’-deoxyguanosine (8-OHdG), and 8-hydroxyguanine. The vitamin D concentration was evaluated using a commercial kit for 25-OH Vitamin D Total ELISA (Gentaur, Sopot, Poland, KAP1971). The total vitamin D measurement was evaluated through the chemiluminescence method using Cobas E411, from Roche company (07464215). The samples and controls were randomized, then measured in the same run, using the blind analysis method.

The oxidative damage marker concentration (8-hydroxy-2′-deoxyguanosine) was measured using a DNA/RNA Oxidative Damage (High Sensitivity) ELISA Kit (Cayman Chemical, Ann Arbor, MI, USA) in the cell culture medium according to the manufacturer’s instructions. The absorbance at a wavelength of 405 nm was read on a Wallac 1420 VICTOR microplate reader (PerkinElmer, Waltham, MA, USA).