Baclight viability dyes

IL-8 treated PMNs in RPMI were allowed to attach to human serum-coated glass coverslips for 1 hr at 37°C, 5% CO₂ as previously described (Johnson et al., 2013b (link)). PMNs were infected with Gc as above, and detection of viable and nonviable Gc associated with PMNs as previously described, using Life Technologies Baclight Viability Dyes (Life Technologies) on saponin-permeabilized PMNs (Johnson et al., 2013a (link)). Images were acquired within 30 min of exposure to the viability dyes. A minimum of 100 bacteria were analyzed for each experiment.

Baclight viability dyes (Life Technologies) were used to stain membrane permeant bacteria (propidium iodide) and total bacteria (Syto9) and analyzed as previously described (Johnson et al., 2013a ). Briefly, acid-washed glass coverslips were coated with 50% normal human serum at 37°C for 30 min prior to infection. Mid-log phase Gc were exposed to neutrophils for 1 hr at 37°C at an MOI of 1. The percent of Gc positive for propidium iodide was calculated relative to total bacteria (propidium iodide-positive and –negative) for the intracellular and extracellular compartments. From these experiments, percent internalization was also determined by dividing total intracellular bacteria by total bacteria, regardless of propidium iodide fluorescence. For experiments assessing serum opsonization and internalization, extracellular bacteria were stained with Alexa Fluor 647-coupled soybean lectin (ThermoFisher), and total bacteria were stained with 5 µM Syto9 (ThermoFisher) following neutrophil permeabilization; percent internalization was determined by dividing the number of intracellular bacteria by the number of cell-associated (internal and external) bacteria. At least 60 bacterial cells were assessed per strain for each independent experiment.

Baclight viability dyes (Invitrogen) were used to assess Gc survival intracellularly and extracellularly in the presence of PMNs as described (Criss et al., 2009 (link)). In some experiments, PMNs were treated with 1x Protease Inhibitor Cocktail Set V containing AEBSF, aprotinin, E-64, and leupeptin (Calbiochem) during attachment to coverslips and throughout the infection.