Cytotox onetm homogeneous membrane integrity assay

THP-1 human monocyte leukemia cell line (ATCC) was grown in RPMI-1640 medium (Gibco, TermoFisher) supplemented with 10% FBS (Sigma-Aldrich), 5,000 U/ml penicillin, 5,000 μg/ml streptomycin, and 200 mM L-glutamine (all from Gibco) at 37°C and 5% CO₂ humidity. The cells were subcultured every second-third day.
MaxiSorp 96-well plates were coated overnight at 4°C with human serum albumin (HSA) or immobilized MFAP4 (both 10 μg/ml). The cells seeded (40.000 cells/well) and differentiated with 5 nM phorbol 12-myristate 13-acetate (PMA; Sigma) for 48 h, equilibrated with complete medium for 24 h, and serum-starved for 48 h. The cells were then stimulated with 20 ng/ml TNF (R&D Systems) for 48 h. Zymography on culture supernatants was performed essentially as described above. Cell proliferation rate was assessed using WST-1 assay (Sigma) according to the manufacturer's instructions. Cell viability was assessed by CytoTox-ONE^TM Homogeneous Membrane Integrity Assay (Promega) according to the manufacturer's instructions. The zymography results were normalized to the cell proliferation index.

Refined storax oil was purchased from Tianjin ZHONGXIN Pharmaceutical Group Limited by Share Ltd Darentang Pharmaceutical Factory and conformed to the standard of China Pharmacopeia (2010 version). Dulbecco’s modified Eagle’s medium/F12 (DMEM/F12), glucose-free DMEM, fetal bovine serum (FBS), HBSS, and 0.25% Trypsin-EDTA were obtained from Gibco (Grand Island, NY, USA). CytoTox-ONETM Homogeneous Membrane Integrity Assay and Cell Counting Kit-8 (CCK-8) were purchased from Promega (Madison, WI, USA) and Dojindo (Kumamoto, Japan), respectively. DCFH-DA and Trizol were obtained from Invitrogen (Eugene, USA). Penicillin and streptomycin were purchased from Beyotime (Shanghai, China). Anti-NF-κB p65, anti-p-IκBα antibody, anti-p-IKK antibody, and anti-β-actin antibody were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-LaminB1 antibody, anti-iNOS antibody, anti-IL-1β antibody, anti-ICAM-1 antibody, and anti-VCAM-1 antibody were purchased from Abcam Technology (Cambridge, MA, USA). SYBR® Select Master Mix and High Capacity cDNA Reverse Transcription Kits were obtained from Applied Biosystems (Foster City, USA).

Following 48 h in serum-free DMEM, cardiomyocytes plated on 12-well tissue plates were subjected to simulated ischemia, induced by replacement of culture media with sterile-filtered Krebs buffer (118 mM NaCl, 4.8 mM KCl, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄, 25 mM NaHCO₃, 50 mM EDTA. 11.0 mM glucose, 1.75 mM CaCl₂), before incubation for 6 h at 37°C under hypoxia (95% N₂-5% CO_2, using a hypoxia chamber, QNA International, Melbourne, VIC, Australia), with subsequent 48 h reoxygenation as described previously (Qin et al., 2017b (link)). Agonists were dissolved in DMEM or Krebs buffer, and were present for the full duration of hypoxia (H) and reoxygenation (R). At the end of 48 h reoxygenation, culture supernatant aliquots were collected on ice and stored at -80°C for assessment of cardiomyocyte injury via levels of lactate dehydrogenase (LDH) released (Cyto-Tox-One^TM Homogeneous Membrane Integrity Assay, Promega Inc, Dane County, WI, United States). Levels of cardiac troponin (cTnI) released from cardiomyocytes were also determined using a high-sensitivity rat cTnI ELISA kit (Life Diagnostics Inc, West Chester, PA, United States) as per manufacturer’s instructions.

Cell membrane integrity was determined using CytoTox-ONE^TM Homogeneous Membrane Integrity Assay (Promega Corporation, USA). Prior to, and after preservation of the cell sheets for 7 days, the supernatants and the cell sheets were collected, and LDH release was quantified. The amount of LDH release was calculated considering an LDH level of 100% for the total of the cell sheet and supernatants.
After preservation for 7 days, the intracellular GSH/GSSG ratio of the hOEC sheet was measured using GSH/GSSG-Glo™ Assay (Promega Corporation, USA) according to the manufacturer protocol. The amount of fluorescence was measured with Multilabel Reader ARVO™X4 (Perkin Elmer).

Lactate dehydrogenase (LDH) in supernatant was measured using CytoTox-ONE^TM Homogeneous Membrane Integrity Assay (Promega) according to the manufacturer’s instructions. LDH release (%) was calculated by using the following formula. LDH release (%) = [(value in sample) − (background)]/[(value in Triton X-100-treated sample) − (background)] × 100.