Lactate dehydrogenase ldh release assay

Human brain microvascular endothelial cells (HBMECs) (Cell systems, Kirkland, WA) were cultured in flasks coated with rat tail collagen I (Corning, Bedford, MA) and maintained in endothelial basal medium (EBM)-2 (Lonza, Hopkinton, MA) supplemented with fetal bovine serum, fibroblast growth factor-2, epidermal endothelial growth factor, hydrocortisone, insulin-like growth factor, ascorbic acid, VEGF, and amphotericin B. Cells of passage 5 to 12 were used for the experiments. When 80–90% confluent, HBMECs were treated with 50ng/ml of recombinant human TNFα (R&D) with or without 1μg/ml of LCN2 (R&D, Minneapolis, MN) for 24hr. Cytotoxicity was measured by a standard lactate dehydrogenase (LDH) release assay (Roche, Germany), and proliferation was evaluated by standard 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyl-tetrazolium bromide (MTT) assay.

Caspase-3 activity was measured from cell lysates according to the manufacturer’s instruction of the EnzChek^® Caspase-3 Assay Kit #2 (Life Technologies) which has been described previously31 (link). The caspase-3 activity was corrected by the total protein level of the cell lysate and normalized to non-treatment (NT) that is set as 100%. Apoptotic cells in primary rat cortical culture were identified by immunocytochemistry using the anti-cleaved caspase-3 antibody (New England Biolabs) followed by Alexa Fluor-568 secondary antibody (Life Technologies), and mounted with Citifluor (London, UK) in the presence of DAPI. Fluorescent images were obtained by the Axioplan epifluorescent microscope (Zeiss, Cambridge, UK), from which positive stained and total number of cells were counted to produce a percentage of apoptotic cells. Lactate dehydrogenase (LDH) release assay (Roche, Burgess Hill, UK) was used to quantify the cell death and the protocols had been described32 (link). The percentage LDH release was obtained from the experimental to the Triton X-100 treated total release. For the detection of neuronal death in primary culture, cells were stained with 0.001% Fluoro-Jade C (FJ-C)(Millipore) after fixed with 4% paraformaldehyde. Positive stained cells were counted under by epifluorescent microscope by examiner blinded to the condition.