Lactate dehydrogenase ldh assay kit

RBC depleted naïve KaLwRij splenocytes were enriched using a CD8a+ T‐cell isolation kit (Miltenyi Biotec), as per manufacturer's instructions. Murine T cells were then cocultured with gamma‐irradiated (30 Gy) 5TGM1 cells at 10:1 effector:target ratio in T‐cell medium (10% FCS RPMI‐1640 medium, 0.05 mM β‐mercaptoethanol, 1× non‐essential amino acids and 2 ng/mL recombinant hIL‐2 (R&D Systems), stimulated with or without hMPO (2 μg/mL). After 72 h, T cells were harvested and seeded at 3 × 10⁴ cells/well with 1 × 10⁴ irradiated 5TGM1 cells in 1% FCS T‐cell medium in a 96‐well plate and cultured for 24 h. Cytotoxicity was determined using a lactate dehydrogenase (LDH) assay kit (Promega Corporation) as per manufacturer's instructions.
For human T‐cell cytotoxicity assays, Vγ9Vδ2 T cells were cultured with or without hMPO (2 μg/mL) for 2 h in 10% heat‐inactivated FCS DMEM. T‐cells were then seeded at 1 × 10⁵/well in a 96‐well plate with 1 × 10⁴ RPMI‐8226 cells and cultured for 4 h. RPMI‐8226 LDH release was determined as outlined above.

To establish the in vitro hepatocyte hypoxia‐reoxygenation (H/R) model, the cells were cultured in serum‐ and glucose‐free DMEM and challenged to a hypoxia state (1% O₂, 5% CO₂, and 94% N₂) in a modular incubator chamber. After 60 minutes of hypoxia, cells were cultured in normoxia situation (air/5% CO₂) with standard DMEM for the indicated time. And then, the cells and medium were collected for further analysis. Lactate dehydrogenase (LDH) Assay Kit (Promega) was used to determine cell cytotoxicity.