Lmax microplate luminometer

Oxidant production was measured by chemiluminescence using 2×10⁵ neutrophils during synchronized phagocytosis of particulate stimuli [20 (link), 21 (link), 38 (link), 39 (link)]. Extracellular ROS was measured using 20μM isoluminol with 20U/ml horseradish peroxidase (HRP), and intracellular ROS was measured using 20μM luminol with 20U/ml HRP and 10μg/ml superoxide dismutase (SOD) [20 (link), 21 (link), 32 (link)]. An Lmax microplate luminometer (Molecular Devices, Sunnyvale, CA, USA) was used to record luminescence as previously described [20 (link), 21 (link)].

This study was approved by Indiana University School of Medicine Institute Review Boards, and written informed consent was obtained from donors. Human neutrophils were isolated from heparin-anticoagulated venous blood using Polymorphprep (AXIS-SHIELD PoC, Oslo, Norwary). Superoxide production by neutrophils stimulated with live or heat-inactivated B. burgdorferi, with or without pyruvate, was measured by luminol chemiluminescence assay [55] (link). Specifically, 2.5×10⁵ human neutrophils were plated into a well of a 96-well plate (COSTAR, Corning, NY) in PBSG (PBS plus 0.9 mM CaCl₂, 0.5 mM MgCl₂, 20 mM dextrose) in the presence of 50 µM luminol and horseradish peroxidase (HRP; final concentration: 20 U ml⁻¹), without or with superoxide dismutase (SOD; final concentration: 75 µg ml⁻¹) and kept on ice for 10 minutes prior to the assay. B. burgdorferi (2.5×10⁶ cells) in 25 µl of PBS were added to wells, and the plate was spun down at 800 r.p.m. for 1 min immediately prior to reading. Relative light units (RLU) were monitored at 60-second intervals for up to one hour by the Long Kinetic module in an Lmax microplate luminometer (Molecular Devices, Sunnyvale, CA). Integrated RLU values were calculated by SOFTmax software (Molecular Devices).

NADPH oxidase activity in intact cells was measured using a modified assay [51] (link). Briefly, photon emission from the chromogenic substrate lucigenin was measured every 15 s for 20 min in an Lmax Microplate Luminometer (Molecular Devices). Lucigenin serves as the acceptor of electron/O₂⁻ generated by the NADPH oxidase complex. Activity measurement was performed in a 250-mM HEPES buffer, pH 7.4, containing NaCl 120 mM, KCl 5.9 mM, MgSO₄ 1.2 mM, CaCl₂ 1.75 mM, EDTA 0.5 mM, glucose 11 mM, lucigenin 0.2 mM, and NADPH 0.1 mM as the substrate. Non-treated or doxorubicin-treated cells were collected, counted, and pelleted at 400×g at 4°C for 4 min; the pellet was re-suspended in a balanced salt solution containing NaCl 130 mM, KCl 5 mM, MgCl₂ 1 mM, CaCl₂ 1.5 mM, phosphoric acid 35 mM, and HEPES 20 mM, pH 7.4. 0.5×10⁶ cells in balanced salt solution containing 10 mM glucose and 1 mg/mL bovine serum albumin were used in measurement. A buffer blank was subtracted from each reading before data calculation. No activity could be measured in the absence of NADPH or in the presence of 100 µM DPI.

Cells were cultured overnight in a 96-well microplate and the next day divided into four groups: untreated, TOX siRNA construct #1, TOX siRNA construct #2, and negative control siRNA. Each treatment was added 24 hours following culture initiation. At 24 hours post-treatment, a CellTiter-Glo^® Luminescent Cell Viability Assay (Promega, Madison, WI) was conducted according to manufacturer’s instructions. Plates were read using the Lmax Microplate Luminometer (Molecular Devices, Sunnyvale, CA) and SoftMax Pro Microplate Data Acquisition & Analysis Software (Molecular Devices). Each experiment was performed in quintuplicate, and statistical analysis comparing viability between treatment groups was conducted using the student’s t-test.

ATP measurements were taken as previously described (48 (link)). After 1 h of treatment, cultures were pelleted, frozen on dry ice, and stored at −80°C until processed. Frozen cell pellets were suspended in 100 µl 0.025 M HEPES buffer with 0.02% Tween 80 (pH 7.75) and added to glass tubes (12 by 75 mm) containing 40 µl chloroform. Mixtures were then heated at 80°C for 20 min, and subsequently, 4.9 ml 0.025 M HEPES buffer (pH 7.75) was added. The ATP assay was performed as per the manufacturer’s protocol (Promega Enliten ATP assay system). Ten-microliter samples were added to a 96-well plate (Costar solid white) and loaded into an L-Max microplate luminometer (Molecular Devices). Enliten luciferase-luciferin reagent dissolved in sample buffer was injected immediately before the results were read. Luminescence was determined every 0.1 s for a total of 100 s. Data are the averages from 6 independent experiments assayed in duplicate.

Cells were plated in triplicate at 2 × 10³ per well in white-walled 96-well plates (Becton Dickinson) for 24 hours and then were treated with the IKK inhibitor and/or cisplatin for additional 48 hours. Caspase-3/7 activity was measured using the Caspase-Glo 3/7 assay (Promega) according to the manufacturer's instructions. Caspase-Glo 3/7 assay uses a caspase-3/7 tetrapeptide DEVD substrate that produces a luminescent signal on cleavage. Relative light units were measured on an Lmax Microplate Luminometer (Molecular Devices).