Ldh assay kit

We exposed the 12d-old cortical neuron-glial co-cultures to RBC-lysate (1μl lysates/100μl medium) in the presence/absence of mrLTF. 20h later, cell viability was assessed with MTT reagent/(G4000/Premega).
To simulate iron-induced neurotoxicity, we added FeCl₃ (1–100μM) to the 12d-old cortical neurons in culture. This produced a dose-dependent neuronal death, with 50μM FeCl₃ causing 95% neuronal death within 24h. To determine the rLTF role in detoxifying iron, we incubated neurons with 10μM FeCl₃ for 6h with or without 20μg/ml of mrLTF. The LDH assay kit (G1780/Promega) was used to measure injury.

LDH sequestration assay was done as described previously (Pattingre et al., 2003 (link)); (Seglen et al., 2015 (link)). Briefly, 3×10⁶ Hela^WT, Stx17^KO or ATG13^KO cells were plated in 10 cm dishes, induced for autophagy using EBSS in presence of Bafilomycin A1 for 2 h. Cells were collected and washed twice with phosphate-buffered saline and then once with homogenization buffer (50 mM potassium phosphate, 1 mM EDTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride (PMSF), 300 mM sucrose, 100 μg/ml bovine serum albumin, and 0.01% Tween 20. Cells were homogenized in 1 ml of cold homogenization buffer by 15 strokes in a glass/Teflon homogenizer on ice, followed by centrifugation at 300 × g for 10 min at 4°C. Post-nuclear material was layere d on 3.5 ml of buffered Nycodenz/ sucrose (10% sucrose, 8% Nycodenz, 1 mM EDTA, 100 μg/ml bovine serum albumin, and 0.01% Tween 20) and centrifuged at 7000 × g for 1 h. The pellet was washed with homogenization buffer and resuspended in buffer containing 2 mM Tris-HCl (pH 7.4), 50 mM mannitol, 1 mM PMSF, 0.5 μg/ml leupeptin, 0.1 μg/ml aprotinin, and 0.7 μg/ml pepstatin. The suspension was sonicated and centrifuged at 15,000 × g for 10 min. The lactate dehydrogenase activity was measured using an LDH assay kit from Promega using the manufacturer’s protocols.