Ldh cytotoxicity assay

Murine aortas were obtained from Balb/c mice. Small sections of human iliac artery were obtained from deceased donor organs. Heparinase I and chemical reagents, unless otherwise indicated, were obtained from Sigma-Aldrich (St. Louis, MO). Alpha-Cyano-4-Hydroxy-Cinnamic Acid (CHCA) MALDI Matrix was from Thermo Scientific (Waltham, MA). Recombinant mouse IL-2 was from Cell Sciences (Canton, MA). The fluorescent dye used to label IL-2 (800CW) was obtained from LI-COR Biosciences (Lincoln, NE). CellTox Green cytotoxicity and CellTiterGlo proliferation assays were from Promega (Madison, WI). LDH cytotoxicity assay was from Roche (Indianapolis, IN). The following antibodies were used: rabbit anti-mouse/human IL-2 receptor (IL-2R) βpolyclonal antibody (Novus Biologicals, Littleton, CO), mouse anti-rat IL-2Rβ monoclonal antibody (clone L316, AbD Serotec, Raleigh, NC), rat anti-mouse blocking monoclonal antibody (clone S4B6, BD Biosciences, San Jose, CA), and a chicken polyclonal antibody recognizing human and murine IL-2 (Sigma). CTLL-2 (mouse cytotoxic T lymphocyte), NRK (normal rat kidney epithelium), HK-2 (human kidney epithelium), B16-F10 (mouse melanoma) cells, and EL4.IL-2 (murine lymphoma) cell lines were obtained from American Tissue Type Collection (Manassas, VA). Smooth Muscle Cell Medium was from ScienCell (Carlsbad, CA).

Freshly collected cell supernatants following stimulation were used for the end-point lactate dehydrogenase (LDH) activity measurement assay according to the manufacturer´s instructions (LDH Cytotoxicity Assay, Roche, Basel, Switzerland). On the cells, we performed XTT test to determine their viability/metabolic activity. For XTT assay, we used DMEM without phenol red and added to it a solution of tetrazolium salt (XTT) and phenazine methosulphate (PMS). Upon colour development we measured absorbance at 490 nm using a multiplate reader SinergyMx (BioTek, Winooski, VT, USA).

Cytotoxicity was analyzed using lactate dehydrogenase (LDH) cytotoxicity assay (Roche, Indianapolis, IN). Briefly, conditioned medium was collected from the respective wells, mixed with the assay solution, incubated for 20 min in the dark, and the absorbance at 490 nm was measured using a spectrophotometer. For a positive control, cells were incubated for 45 min at 37 °C with 1% Triton X (TX)-100.

Cytotoxicity was analyzed using the LDH cytotoxicity assay (Roche). Briefly, conditioned medium was collected from the respective wells, mixed with the assay solution, incubated for 20 min in the dark, and the absorbance at 490 nm was measured using a spectrophotometer. For a positive control, cells were incubated for 45 min at 37 °C with 1% Triton X-100.

A549 respiratory epithelial cells (obtained from ATCC, catalog number CCL-185) were grown in minimum essential medium (MEM) with 10% fetal bovine serum as described [20 (link)]. Cell monolayers (80–90% confluent in sterile 24-well plates) were incubated in MEM without serum overnight prior to stimulation with ~10⁸ cfu of the indicated S. pneumoniae strains for 12 hrs. LDH cytotoxicity assay (Roche) was performed as per the manufacturer’s instructions, and values were normalized to 0% (vehicle control) and 100% lysis (1% Triton X-100) controls as previously described [21 (link)].

Decidual tissue was washed in sterile phosphate-buffered saline, cut into pieces (explants with wet weight range of 15–33 mg) and distributed in the culture plate evenly between the different culture conditions (24 ± 2 mg, mean ± standard deviation). There were no significant differences in explant weight between the culture conditions. Explants were cultured in Ham's F12/Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum and 100 mg/mL penicillin-streptomycin (Sigma-Aldrich) and incubated for 24 h at 37°C, 8% O₂ and 5% CO₂ (33 (link)). Culture medium was then replaced by fresh culture medium with or without 500 pg/ml LPS priming (#tlrl-3pelps, InvivoGen, California, United States). After 2 h, the medium was replaced by fresh culture medium with or without stimuli; 200 or 2,000 μg/ml synthetic cholesterol crystals (#C3045, Sigma-Aldrich) or the positive control 3 mM ATP (#A7699, Sigma-Aldrich). Supernatants were collected after 24 h, centrifuged and stored at −80°C. Six technical replicates for each experimental condition were combined before analysis. Tissue viability was assessed by lactate dehydrogenase (LDH) cytotoxicity assay (#04744926001, Roche, Basel, Switzerland) (Supplementary Figure 1). IL-1β levels in supernatants were measured undiluted in duplicate using quantitative sandwich ELISA (#557953, BD Biosciences, New Jersey, United States).