Cyquant ldh cytotoxicity assay kit

Levels of lactate dehydrogenase (LDH) were measured by CyQUANT^TM LDH cytotoxicity assay kit (Invitrogen) as an indicator of cytotoxicity, following manufacturing protocol. Briefly, human monocytic THP-1 cells (ATCC) were cultured and differentiated into macrophages as described above. After being treated with different tau species of 500 nM monomeric equivalent concentration for 24 h, 50 μL of conditioned media of each sample along with maximum LDH activity controls (generated using 10× lysis buffer) and spontaneous LDH activity controls (no treatment group) were incubated with 50 μL LDH reaction mixture in a 96-well flat-bottom plate at room temperature for 30 min. Afterward, 50 μL of stop solution was added to each sample well to quench the reaction. Absorbance was measured at 490 nm and 680 nm: before calculating the percentage of toxicity, the 680 nm absorbance value (background) must be subtracted from 490 nm absorbance, and then the percentage of cytotoxicity can be determined using the following formula: $\mathrm{Percent}\mathrm{of}\mathrm{cytotoxicity}=\frac{{\mathrm{LDH}}_{\mathrm{sample}}-{\mathrm{LDH}}_{\mathrm{spontaneous}}}{{\mathrm{LDH}}_{\mathrm{maximum}}-{\mathrm{LDH}}_{\mathrm{spontaneous}}}$

Cytotoxic cell damage was determined using the CyQUANT^TM LDH Cytotoxicity Assay Kit (Invitrogen), which measures cytotoxicity based on extracellular lactate dehydrogenase (LDH) activity, according to manufacturer’s guidelines. Briefly, media was collected at the end of each experiment and centrifuged to remove any remaining cells or debris, and the supernatant was collected and used for the assay. 50 μL of the medium was combined with 50 μL of the reaction mixture in a flat-bottomed, 96-well plate and incubated for 30 min at room temperature in darkness. The reaction was terminated with 50 μL of stop solution and absorbance at 680 nm measured and subtracted from absorbance at 490 nm.

Following overnight stimulation, the supernatant from each well was transferred to microcentrifuge tubes and span at 1000× g for 10 min. Interleukin-8 (IL-8; marker of inflammation) was measured using Enzyme Linked Immunosorbent Assay (ELISA; Human IL-8/CXCL8 DuoSet; R&D Systems, Minneapolis, MN, USA) within the detection range of 31.2 to 2000 pg mL⁻¹. The absorbance of each well at 450 nm was measured by a microplate reader (SpectraMax i3x, Molecular Devices, San Jose, CA, USA). Cytotoxicity was examined by measuring the release of lactate dehydrogenase (LDH) from the cell supernatants using the CyQUANT^TM LDH Cytotoxicity Assay kit (Invitrogen, Waltham, MA, USA). The absorbance of each well at 490 nm was measured. Percent cytotoxicity was calculated as: (Treatment LDH activity − Control LDH activity)/(Maximum LDH activity − Control LDH activity) × 100.

H9c2 cells were plated onto 96-well plates at a concentration of 5000 cells/well. The next day, the medium was changed to the H9c2 growing medium supplemented with 10% exosome depleted FBS (System Biosciences, Palo Alto, CA, USA) instead of the regular FBS (EV-free medium). Then, the cells were treated with 10 × 10⁹ or 20 × 10⁹ particles/mL of serum EVs derived from saline-treated rats (SAL_EVs) or serum EVs derived from DOXO-treated rats (DOXO_EVs). The cells were incubated with EVs for 24 h. After that, the cells were washed with PBS and treated with 100 or 200 μM hydrogen peroxide (H₂O₂) in EV-free medium for 24 h. The maximum LDH release was induced by adding 10 μL of 10X Lysis Buffer to the cells plated at the same concentration without any treatment and incubated the cells for 45 min in an incubator at 37 °C. After incubation, cell death was evaluated using a CyQUANT^TM LDH Cytotoxicity Assay Kit (Invitrogen, Waltham, MA, USA). Briefly, the supernatants of the treated cells were collected and mixed with the reaction mixture. After incubation for 30 min, a stop solution was applied. The absorbance at 490 and 680 nm was measured. Percentage of cytotoxicity was calculated using the following formula: % cytotoxicity = [ (H₂O₂-treated LDH activity—spontaneous LDH activity)/(maximum LDH activity—spontaneous LDH activity)] × 100.

CyQUANT^TM LDH Cytotoxicity Assay Kit (Invitrogen Corporation, New York, NY, USA) was used to detect cytotoxicity, as lactate dehydrogenase (LDH) release is an indicator of cell cytotoxicity [41 (link)]. Every reagent was provided in the kit. A2058 cells (7.5 × 10⁵ cells/mL) were seeded in a transparent 96-well plate. After 72 h treatment, 50 μL media of each sample was transferred to a new transparent 96-well plate. For 30 min, the wells were incubated with 50 μL Reaction Mixture, then the reaction was stopped by adding 50 μL Stop Solution to each sample well and absorbance was obtained at 490 nm by a plate reader (Multiskan MS, Labsystems, Helsinki, Finland). The experiment was performed in triplicates, and the results were normalized to the untreated medium control and represented as mean ± SD.

Donor T cells were extracted from spleens of mice with aGvHD (day 7 post transplant) or control C57Bl/6 mice by negative isolation (STEMCELL Technologies) as described above. Cells were plated in media at 4 × 10⁵ cells/well in 96 well round-bottom tissue culture plates. After 4 h, supernatant was collected and used for LDH or cytokine quantification. LDH was measured using the CyQUANT^TM LDH Cytotoxicity Assay Kit (Invitrogen) and IL-1β and IL-18 levels were measured by ELISA (DuoSet Mouse ELISAs, R&D Systems). Mouse T cells isolated from C57Bl/6 mice were activated using Dynabeads Mouse T-Activator CD3/CD28 (Gibco), according to the manufacturer’s protocol, in media with IL-2. After 48 h, cells were collected and lysed for western blot analysis of caspase-1 expression.

The cytotoxic effect of BaP in U1 and SVGA cells was evaluated by LDH release using the CyQUANT™ LDH Cytotoxicity Assay Kit (Catalog no. C20300, ThermoFisher Scientific) according to the manufacturer’s instruction. Briefly, U1 and SVGA cells were exposed to BaP (0.01–1 μM) for 24, 48, and 72 h. Cell culture supernatants were collected at indicated time points and transferred to a 96-well plate. The reaction mixture was added to the cell culture supernatant for 30 min at room temperature. Background absorbance 680 nm was subtracted from 490 nm absorbance to determine the LDH activity.