The reduction of intracellular LDH and its release into the extracellular medium is a sensitive indicator of nonreversible cell death due to the damaged caused to the cell membrane caused by cell apoptosis or necrosis (Yang et al. 2018 ). One hundred microliters of cell culture medium supernatant was collected to determine LDH activity. The test was performed according to the LDH assay kit manufacturer’s instructions (Nanjing Jiancheng, China). As described above, after AMs were treated for 4 h with PM2.5 (at 0, 33, 100, or 300 μg/mL), a 100 μL aliquot of cell culture medium supernatant was collected from each dish (105 cells) in order to test extracellular LDH activity. Briefly, 250 μL of the reconstituted substrate mix and 50 μL Coenzyme I solution (Nanjing Jiancheng LDH assay kit) were added to each sample; following incubation in a 37 °C water bath for 15 min, the enzymatic reaction was stopped with 25 μL of 2,4-dinitrohydrazine (Nanjing Jiancheng LDH assay kit). The mixture was incubated again for 15 min in a 37 °C water bath and 2.5 mL NaOH (0.4 mol/L) was added. The absorbance was then measured at 440 nm using an ultraviolet (UV)-visible spectrophotometer (Beckman DU-640B, USA). All experiments were performed in triplicates.
Ldh assay kit
Manufactured by Nanjing Jiancheng
Sourced in China
The LDH assay kit is a laboratory equipment used to measure the activity of the enzyme lactate dehydrogenase (LDH) in a sample. LDH is an important enzyme involved in various metabolic processes and its levels can be used as a biomarker for certain medical conditions. The kit provides reagents and a protocol to quantify LDH levels colorimetrically or fluorometrically.
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Lab products found in correlation
Sod assay kit, by Nanjing Jiancheng (9 mentions)
Microplate reader, by Thermo Fisher Scientific (8 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (6 mentions)
Ck mb assay kit, by Nanjing Jiancheng (5 mentions)
Microplate reader, by Bio-Rad (5 mentions)
Lactate assay kit, by Nanjing Jiancheng (5 mentions)
Mda assay kit, by Nanjing Jiancheng (4 mentions)
Cck 8 assay kit, by Dojindo Laboratories (4 mentions)
Cck 8, by Nanjing Jiancheng (3 mentions)
Cell counting kit 8 cck 8 kit, by Beyotime (3 mentions)
Ck assay kit, by Nanjing Jiancheng (3 mentions)
T aoc assay kit, by Nanjing Jiancheng (2 mentions)
Ros kit, by Nanjing Jiancheng (2 mentions)
Dmem low glucose media, by Thermo Fisher Scientific (2 mentions)
Ultra micro total atpase kit, by Nanjing Jiancheng (2 mentions)
Atp content assay kit, by Nanjing Jiancheng (2 mentions)
Malondialdehyde mda assay kit, by Nanjing Jiancheng (2 mentions)
Cell counting kit 8 cck 8, by Dojindo Laboratories (2 mentions)
Microplate reader, by Agilent Technologies (2 mentions)
Infinite m1000 pro, by Tecan (2 mentions)
218 protocols using ldh assay kit
1
LDH Assay for Cytotoxicity Evaluation
2
Astragaloside IV Protects HUVECs from Ox-LDL
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Human umbilical vein endothelial cells (HUVECs) were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Astragaloside IV (purity >98%, Cat. No. 84687-43-4) was purchased from Nanjing Spring & Autumn Biological Engineering Co. (Nanjing, China), dissolved in DMSO, and frozen at −80°C. Ox-LDL (oxidized low-density lipoprotein) was purchased from Beijing Solarbio Life Science Company (Cat. No. H7950, Beijing, China). Low-glucose Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS) and AnnexinV-FITC kits were purchased from Invitrogen (Cat. No. V13241, Carlsbad, CA). Cell counting kit-8 (CCK-8) kits were obtained from Beyotime Institute of Biotechnology (Cat. No. C0037, Shanghai, China). LDH assay kits were purchased from Nanjing Jiancheng Bioengineering Institute (Cat. No. A020-1, Nanjing, China). The Transwell chambers (8-μm diameter, 24 wells) were purchased from Corning, Inc. (Corning, NY). Matrigel matrix was purchased from BD System (Cat. No. 356234, Franklin Lakes, NJ). DCFH-DA (2′,7′-dichlorofluorescin diacetate) was obtained from Sigma Chemical Co. (Cat. No. D6883, St. Louis, MO). NADPH oxidase kits were purchased from Genmed Scientific, Inc. (Cat. No. GMS50067, Shanghai, China). ELISA kits for TNFα (Cat. No. 88-7346-77) and IL-6 (Cat. No. 88-7066-77) were purchased from Invitrogen (Carlsbad, CA).
3
Cardiac Biomarker Detection Protocols
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CK-MB and cTnI levels were detected with the help of CK-MB ELISA kits (KE1677, Immunoway) and cTnI ELISA kits (KE1457, Immunoway) following the manufacturers’ instructions, respectively [22 (link),26 (link)]. Additionally, LDH levels were determined by colorimetry using LDH assay kits (A020-1-1, Jiancheng).
4
Antioxidant Evaluation of Defatted Tree Peony Seed Flour
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Defatted tree peony seed flour was provided by the Huarui Science and Technology Development Co. Ltd. (Heze, Shandong, China). Soybean oil was purchased from a local supermarket in Jinan, Shandong, China. Alcalase 2.4 L (2.0 × 105 U/g) was purchased from Novo Co., Ltd. (Beijing, China). The 1-anilino-8-naphthalene-sulphonate (ANS), DPPH, chloroform, tween-40, β-carotene, linoleic acid, glutathione (GSH), and 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) were purchased from Sigma Co., Ltd. (St. Louis, MO, USA). Dulbecco’s modified Eagle medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco (Grand Island, NY, USA). Penicillin–streptomycin, 0.25% trypsin-EDTA, and trichloroacetic acid (TCA) were purchased from Beijing Solarbio Science Technology Co., Ltd. (Beijing, China). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylthiazolium bromide (MTT) and LDH assay kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). SOD, MDA, CAT, and Bicinchoninic acid (BCA) assay kits were purchased from Beyotime Institute of Biotechnology (Shanghai, China). All other chemicals and reagents used were of analytical grade.
5
Cardiac Enzyme Levels After Reperfusion
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After 120 minutes of reperfusion, arterial blood was collected from the right ventricle of the rats. The supernatant was collected after centrifugation using a C2500-R-230V centrifuge (Labnet, Edison, NJ, USA). Serum CK-MB and LDH levels were determined by enzyme-linked immunosorbent assay (ELISA) using CK-MB assay kits (Elabscience Biotechnology, Wuhan, China) and LDH assay kits (Nanjing Jiancheng, Nanjing, China).
6
Quantifying Cell Cytotoxicity: LDH Assay
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To supplement the MTT assay, we used LDH assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) to measure the LDH released from cells. After the cells had been exposed to different treatments, the total culture medium was collected and the amount of LDH released by the cells was determined in accordance with the manufacturer’s protocol. The absorbance values at 440 nm of samples were measured by an automatic microplate reader (CORONA SH-1000 Lab, Japan).
7
Enzymatic Activity Quantification Protocol
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The activities of SOD and LDH were detected using SOD (cat. no. A001-3-2; Nanjing Jiancheng Bio-engineering Institute Co., Ltd.) and LDH assay kits (cat. no. A020-2; Nanjing Jiancheng Bio-engineering Institute Co., Ltd.). The protein extracts were incubated with working solutions according to the manufacturer's protocols and then the absorbance was measured with a multimode reader at 405 nm.
8
Cytotoxicity Evaluation Using CCK-8 and LDH Assays
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Cytotoxicity was assessed by measurement of cell viability and LDH release in the medium using CCK-8 and LDH assay kits, respectively (Nanjing Jiancheng Bioengineering Institute, China). For CCK-8 assay, cells in 96-well plates were treated according to the experimental procedure and then incubated with 10% CCK-8 reagents and 90% fresh DMEM media for 60 min at 37°C before the assay was performed. Media in 6-well plates were centrifugalized to gather supernatant for LDH release test. All operations were carried out according to the manufacturer's instructions.
9
Cytotoxicity Evaluation in Cardiomyocytes
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Cell viability and LDH release were measured to evaluate the extent of cellular injury in primary cardiomyocytes and H9C2 cells using CCK-8 and LDH assay kits, respectively (Nanjing Jiancheng Bioengineering Institute, China) according to the manufacturer's instructions.
10
Bronchoalveolar Lavage Fluid Analysis
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After the instillation of vehicle solution or CuONPs suspension, BALFs were obtained from the trachea by rinsing the lungs three times with warmed 0.9% sterile physiological saline. Then the BALFs were centrifuged at 2500 rpm for 10 min at 4 ℃. The pellets were collected and re-suspended with PBS, the cell counts in BALFs were further determined by TC20TM Automated Cell Counter (Bio-Rad,Hercules, USA). The concentrations of protein in the supernatant without cells were quantified by BCA kits (Bio-Rad). Lactate dehydrogenase (LDH) activity in BALFs were detected by LDH assay kits according to the manufacturer’s protocol (NanJingJianCheng, Nanjing, China).
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