Cyquant lactate dehydrogenase ldh cytotoxicity assay

Analysis of the cell culture media following HD exposure was performed using the CyQUANT Lactate Dehydrogenase (LDH) Cytotoxicity Assay (Invitrogen; Waltham, MA, USA) to determine the presence of necrotic cell death in the exposed tissues [30 (link)]. The assay was performed according to the manufacturer’s specified protocol. Experimental samples were read on a SpectraMax Plate Reader and the results were expressed as a percentage of relative cytotoxicity compared to untreated control.

The CyQUANT™ lactate dehydrogenase (LDH) Cytotoxicity Assay (Invitrogen™) was used to examine treatment-related cytotoxicity. The cerebrovasculature was extracted and treated as above. After being collected in 500 μl of PBS the capillary fraction was divided into three aliquots, one for the maximum LDH release, one for the vehicle treatment induced LDH release (spontaneous LDH release) and one for the treatment induced LDH release. For the maximum LDH release the capillary fraction was resuspended 160 μl PBS with 16 μl of 10× lysis buffer and incubated at 37°C for 45 min as per the manufacturer’s instruction. The supernatant was then collected and put on ice until measurement. The vehicle and treated fractions tubes were resuspended in the vehicle and treated solution each and incubated at 37°C for 20 min. The supernatants were collected and put on ice until measurement. When all samples were ready 50 μl of each were plated in duplicate into a 96-well plate along with a positive control. A 15% CV cut-off was used. The % Cytotoxicity was calculated using the following formula:

Cytotoxicity assays were performed using the CyQUANT™ Lactate Dehydrogenase (LDH) Cytotoxicity Assay (Invitrogen) according to the manufacturer’s recommendation. Cells were seeded in 96-well tissue culture plates in triplicate in STEMdiffperformed using the Neural Progenitor Medium (STEMCELL Technologies) at a density of 2.5 × 10⁴ cells per well. Besides compound-treated samples, two assay controls were added: untreated cells (spontaneous LDH activity) and lysed cells (maximum LDH activity). Fifty microliters of medium from each sample was transferred to a flat bottom 96-well plate and supplemented with 50 μl of LDH Cytotoxicity assay reaction mixture (containing assay substrate in assay buffer prepared according to the manufacturer’s recommendations), followed by 30 min incubation in the dark. Subsequently, 50 μl of Stop Solution was added before absorbance measurements at 490 and 680 nm were taken using a FLUOstar Omega plate reader (BMG Labtech). The A680 nm was subtracted from A490 nm as background before the calculations. Relative cytotoxicity was expressed as follows: