Toxilight bioassay kit

Cells were cultured on uncoated 6-well plates until confluent in DMEM containing 10% FBS, then were switched to DMEM containing 1% FBS. After 1 week, cells were treated with 2 mL DMEM containing implant levels of bimatoprost (10–1000 µM) or vehicle for 24 hours. As a positive control, one well from 3 different TM cell strains was treated with 1% Triton X-100 to lyse all of the cells 30 minutes prior to media collection. Media was collected from all wells and frozen at −80°C. ToxiLight bioassay kit (Lonza) was used to measure the release of adenylate kinase (AK) from damaged cells. Frozen conditioned media, AK detection agent, and AK buffer were brought to room temperature. Detection agent was added to buffer (10 mL), mixed, and incubated at room temperature. After 15 minutes, 100 µL of mixture was added to 20 µL of conditioned media from samples (in triplicate) in a 96-well white/opaque bottom plate while maintained in dark conditions. After 5 minutes, plate luminescence was measured.

Oli-neu cells were seeded in poly-D-lysine coated 96-well plates (Corning) and transfected on the same day with the different miRNA mimics or negative control. After 72 h, cell proliferation was measured using the Cell Proliferation Kit II (Roche), incubating the cells with the XTT reagent for 4 h. Absorbance was finally read at 492 nm with a VersaMax plate reader (Molecular Devices). For cell toxicity measures, an aliquot of conditioned media (20 μL) was removed before adding the XTT reagent and the amount of adenylate kinase released by damaged cells was quantified with the ToxiLight Bioassay Kit (Lonza), according to the manufacturer’s instructions. Luminescent signals were read with a Gemini plate reader (Molecular Devices). Three independent experiments were carried out, running each miRNA mimic transfection in triplicate.

The adenylate kinase assay (ToxiLight bioassay kit LT07-217; Lonza) to assess toxicity was performed following the instructions of the manufacturer. Briefly, 5 μl of the medium where cells were cultured was transferred to a 384 Greiner LUMITRACTM white plate and allowed to reach room temperature. 25 μl of the adenylate kinase detection reagent were then added to each well and incubated for 5 min. Finally, the plate was read in the PHERAstar FSX microplate reader (BMG LABTECH).

T cell clones were co-cultured with 100,000 indicated target cells at indicated ratios at 37°C, with or without CD3/CD28 activation (T-Cell TransAct polymeric nanomatrix inducing low to no death of activated T cells). Supernatants were harvested 18h later and cell death was measured using a bioluminescent, non-destructive cytolysis assay kit designed to measure the release of the adenylate kinase from damaged cells (ToxiLight™ BioAssay Kit, Lonza). The ToxiLight Lysis Control Set (Lonza) was used to determine 100% lysis. Lysis percentages were calculated as follows:
((Luminescence read - Luminescence of target cells alone lysed spontaneously x 100)/(Luminescence of totally lysed target cells – (Luminescence of target cells alone lysed spontaneously + Luminescence of corresponding T cells alone lysed spontaneously)