Ldh glo cytotoxicity assay

After preparation of the Caco-2 monolayers and administration of the infected THP-1 macrophage supernatant, we used the LDH-Glo™ Cytotoxicity Assay and the NADP/NADPH-Glo™ Assay (Promega, Madison, WI, USA) to quantify plasma membrane damage and oxidative stress, respectively. Briefly, for the former assay, all components were combined in warm media and administered to intact, adherent cells inside their respective wells. The cells were incubated for 30 min, and luminescence was measured with the GloMax Navigator™ (Promega, Madison, WI, USA) For the latter assay, cells were lysed with 0.2 N NaOH solution with 1% DTAB, and the lysate was separated to be subjected to heat under acidic or basic conditions to decompose NADPH or NADP⁺, respectively, according to manufacturer protocols. The heated lysate was then cooled to room temperature and administered the assay components. After a 30 min incubation, luminescence was again quantified with the GloMax™ Navigator. Cells were not reused between assays. All experimental groups were plated and assayed in triplicate.

CellEvent™ Caspase-3/7 Detection Reagents (Invitrogen, C10423) were applied to image apoptotic nuclei and detect toxicity in short- and long-term treatment conditions on D7, D9, D11, D14 and D28. Positive controls were pre-treated for 6 h with 1 µM Staurosporine, an apoptosis inducer, in EGM2 MV + 1% AGS tri-culture media. Then, 1× staining solution at 5 µM in tri-culture media was applied to the reservoirs of the microfluidic devices, and incubated for 60 min at 37 °C. Afterwards, cells were fixed, co-stained with UEA I, PDGFRβ and DAPI to determine co-localization of apoptotic nuclei with specific cell types.
LDH-Glo™ Cytotoxicity Assay (Promega, J2380) was used to detect LDH release in supernatants collected on D7, D9, D11, D14 and D28. Positive controls for maximum LDH release were treated with 0.9% Triton X-100 on D7 and D28. Supernatants were diluted 1:100 in LDH storage buffer (200 mM Tris-HCl (pH 7.3), 10% Glycerol, 1% BSA) and stored at -20 °C. These samples were then mixed 1:1 with LDH detection reagent and luminescence was measured after 60 min incubation at RT.

The supernatants of the cell culture experiments were examined to detect the released lactate dehydrogenase (LDH) at the end of the incubation period using the LDH-Glo™ Cytotoxicity Assay (Promega J2380) according to the manufacturer’s recommendations. The VBF samples were additionally measured before incubation, to determine their intrinsic LDH amount.

Evaluation of cytotoxicity was performed by incubating A431 cells with PBMCs (effector-to-target ratio 10:1) in 96-well tissue culture plates with the desired antibody concentration for 24 – 48 h. After different time points, the supernatant was used to measure released LDH by the LDH-Glo Cytotoxicity Assay (Promega) following the manufacturer’s instructions. Plates were observed under a bright-field microscope to evaluate potential cell killing. Further, dead cell staining was performed by trypsinising and collecting all cells, staining with propidium iodide (PI) (Sigma Aldrich) and measuring PE fluorescence using a flow cytometer.

Cell death was assessed using propidium
iodide (PI) staining and the LDH-Glo Cytotoxicity Assay (Promega,
Cat #J2380). For PI staining, MSCs were seeded at 61,000 cells/well
in a 6-well plate. The MSCs were cultured for 48 h in 5 mL of 0.5%
FBS media with a DMSO control (1 μL/mL) or 0.5% FBS media with
1, 5, 10, 20, and 25 μM of PCB mixtures dissolved in DMSO. The
cells were then lifted and stained with PI (Sigma-Aldrich, Cat # P4864)
to a final concentration of 0.01 μM. Controls included unstained
MSCs as well as dead control cells which had been permeabilized with
0.2% TritonX-100 for 10 min. The cells were then incubated in the
staining solution for 10 min before being analyzed via flow cytometry
using a Cytek Northern Lights spectral cytometer. For the LDH assay,
MSCs were plated in a 24-well plate as described in the “Cell
Proliferation and Morphology” section. After 48 h of incubation,
2 μL of media was collected from each well, and each sample
was diluted with 48 μL of the LDH storage buffer. The LDH detection
reagent was prepared and added to each sample as directed by the manufacturer’s
protocol. After incubation for 1 h at room temperature, luminescence
was recorded and normalized by the positive control to obtain LDH
release as a percentage of the dead cell control.

For viability analysis, PCLS culture medium was collected and centrifuged (8 min, 640 g). Supernatant was transferred to a new tube and diluted 1/5 in lactate dehydrogenase (LDH) storage buffer (200 mM Tris–HCl, 10% glycerol, 1% BSA) and stored at −80°C until analysis. LDH levels were measured using the LDH-Glo Cytotoxicity Assay (Promega) according to manufacturer’s instructions.

To investigate the direct impact of the biomaterial on the L-929 cell line, cells were seeded into 6-well plates pre-coated with GO-supplemented biomaterials (HGO1, HGO2, BGO1 and BGO2) at densities of 2.5 × 10⁵/well, 6.3 × 10⁴/well and 4 × 10³/well. The cells were subsequently incubated at 37 °C and in a humidified 5% CO₂ atmosphere in a DMEM medium for 1, 3 and 7 days.
The LDH-Glo™ Cytotoxicity Assay (Promega, Madison, WI, USA) was used to evaluate cytotoxicity resulting from cell interactions with biomaterials using the L-929 cell line (ATCC^®, CCL-1™, Manassas, VA, USA). The assay was conducted at three distinct time points (1, 3, and 7 days).
To carry out the assay, the culture medium was collected and diluted at a ratio of 1:100 in the LDH Storage Buffer. The samples were stored at −20 °C until testing. The entire procedure had been carried out according to the manufacturer’s protocol. The luminescence signal was measured using a microplate reader (BioTek Synergy H1 Plate Reader) after a one-hour incubation at room temperature.