Cytotoxicity detection kitplus ldh kit

HEK293T and MN-1 (mouse motor neuron–neuroblastoma fusion) cells were cotransfected with pcDNA3-mCherry and pcDNA3.1-TRPV4-FLAG (WT or mutant) constructs using Lipofectamine LTX. HC-067047 (5 μM) was added 4.5 hours after transfection. Calcium imaging was performed on a Zeiss Axio Observer.Z1 inverted microscope equipped with a Lambda DG-4 (Sutter Instrument Company, Novato, CA) wavelength switcher. Cells were bath-loaded with Fura-2 AM (8 μM; Life Technologies) for 30 minutes at 37°C in calcium-imaging buffer (150 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 2 mM CaCl₂, 10 mM glucose, 10 mM HEPES, pH 7.4) containing 0.04% Pluronic F-127. Transfected cells were identified for analysis by mCherry expression. Cell death assays were performed 24 hours after transfection using the Cytotoxicity Detection Kit^PLUS (LDH) kit (Roche Diagnostics, Indianapolis, IN) according to the manufacturer's instructions. Cytotoxicity in this assay is calculated by subtracting the absorbance of a background control (normal medium) from the absorbance of the experimental samples (supernatant from transfected cells). Results are expressed as a percentage relative to the absorbance of a high control (supernatant from cells treated with lysis buffer). Statistical significance was determined using the Mann-Whitney U test (calcium imaging) or unpaired Student t test (cytotoxicity assays).