Cyquant ldh cytotoxicity assay

Cytotoxicity was measured using the CyQUANT LDH Cytotoxicity Assay (Invitrogen). H508 cells were cultured at 50% confluence on 96-well flat-bottom plates. After serum starvation overnight, cells were treated with test chemicals for five days. The assay was performed per the manufacturer’s instructions; briefly, 50 µL of sample medium was removed from each well and treated with the reaction mixture followed by a 30-min incubation at room temperature. After 30 min, stop solution was added and the plates were examined using a microplate reader set at 490 and 680 nm. Percent cytotoxicity was determined using the manufacturer’s instructions.

The toxicity of the pIB/V5-His-based effector library was measured using a CyQUANT LDH Cytotoxicity assay (Invitrogen; S4 Table). Briefly, 10,000 LD652 cells per well were plated into 96-well plates and transfected the next day with the pIB/V5-His-based library (50 ng of DNA per well). Following 48 h of expression, 25 μL of supernatant media was transferred to a fresh 96-well plate and 25 μL of reaction mixtures was added to each well. The plates were mixed gently by tapping ~5 times and incubated in the dark for 30 min, then 25 μL of Stop Solution was added. The absorbance of each plate was read at both 490 nm and 680 nm within 15 min of adding Stop Solution. Data plotted indicates the 680 nm value (background signal of instrument) subtracted by the 490 nm value, per manufacturer’s instructions. Controls included: an LDH positive control provided with the kit, LD652 “media-only” negative control, and an “untransfected” cell control, and a”lysed moth cells” positive control that indicates where untransfected LD652 cells were lysed per manufacturer’s instructions 1 h before collecting supernatant media.

BMDM, MLE-12, and BAL cell culture supernatants were collected after treatment with M1 or LPS for 20 h for ELISA and after 0–48 h for LDH and ROS assays. BAL fluid samples were obtained from the mice, and cytokines were measured using IL-6, CXCL1, CXCL-10, CCL2, CCL5 ELISA kits (all from R&D Systems, Minneapolis, Minnesota, USA). LDH and ROS were detected using the CyQUANT™ LDH cytotoxicity assay (Invitrogen) and in vitro ROS/RNS assay kits (Cell Biolabs Inc. San Diego, CA, US), respectively, according to the manufacturers’ protocols.

HEp-2 cells were seeded at a density of 2000 cells/well in 96 well plates (Falcon, 353072) in DMEM, 5% FBS and 2 mM l-Gln. Cells were treated with Ssp for 5 h or 24 h. CyQUANT LDH Cytotoxicity Assay (Invitrogen, C20301) was used to determine LDH activity according to manufacturer’s instructions.

Isolated NK cells were plated at 100,000 cells/well, in 50uL of medium, with or without IL-15 supplementation (10 ng/mL), as indicated, for 1h at 37°C in 5% CO₂. After 1h, K562 cells were co-cultured with NK cells, at an effector-target range of 10:1, in a final volume of 100uL, for 4h at 37°C in 5% CO₂. Target cell killing was analyzed by measuring LDH concentration in the culture supernatant, using the kit CyQuant LDH Cytotoxicity Assay (Invitrogen) following the manufacturer’s instructions. Cytotoxic activities were calculated using the following formula: $\%cytotoxicity=\frac{\left(LDHKilling-LDHNKSpontaneous-LDHK562Spontaneous\right)}{\left(LDHK562\mathit{Max}-LDHK562Spontaneous\right)}\times 100$

miR-501^ΔMP myofibers were treated with CTX for 1 hour at the final concentration of 0.2 μM. LDH release was assessed using the CyQUANT LDH Cytotoxicity Assay (Invitrogen). 50 μl of cell culture media were incubated with 50 μl of Reaction Mixture for 30 min at room temperature, followed by the addition of 50 μl of Stop Solution. Absorbance was measured at 490 nm and background values of 680 nm were subtracted. Data were normalized to total protein content assessed using the Pierce BCA Protein Assay Kit (Thermo Sientific) and expressed as fold change vs control.