Lb953 luminometer

The generation of ROS by the phagocytes, AM and PMN, present in the BAL was determined by a luminol-dependent chemiluminescence assay (Roberts et al. 2014 (link)). The contribution of AM and PMN in the production of ROS was determined using the stimulants, phorbol 12-myristate 13-acetate (PMA) – stimulant of AM and PMN, and non-opsonized, insoluble zymosan – a stimulant of AM only. A resuspension of the BAL cells, equivalent to 5×10⁵ total BAL cells or 5×10⁵AM, was incubated with the luminol for 10 min at 37 °C which was followed by stimulation of the cells with either 10 μM PMA or 1 μg zymosan, respectively, in a total volume of 500 ml. The oxidant production by the BAL cells in the absence of the stimulants was measured and considered as the baseline. A Berthold LB 953 luminometer (Wildbad, Germany) was used for the measurement of CL for 15 min at 37° C, and the integral of counts per minute (cpm) per one million cells versus time was calculated. The cpm of the stimulated cells minus the cpm of the corresponding resting cells was calculated and the value was normalized to the total number of BAL cells for PMA-stimulated CL and total number of AM for zymosan-stimulated CL.

Luminol-dependent CL was completed to measure macrophage activity and was monitored using a Berthold LB953 Luminometer (Berthold, Wildbad, Germany). BAL cell (1 × 10⁶ AM/ml) luminescence was measured before and after zymosan stimulation (2 mg/ml; Sigma Chemical Company, St Louis, MO). zymosan was used for its ability to stimulate and be readily engulfed by activated phagocytic cells (Gantner et al., 2003 (link)). Results are presented as zymosan-stimulated minus unstimulated cell CL production.
A 5,5′-dimethylpryrroline N-oxide (DMPO) spin trap technique was implemented to form long-lived free radicals that could be detected via EPR to assess the reactivity of the cells. EPR measurements were collected using a flat cell assembly and Bruker EMX spectrometer (Billerica, MA). Second BAL cells (1 × 10⁶ AM/ml) were combined with 200 mM DMPO (Sigma Aldrich), and 2 mM Cr (VI) for 3 min at 37 °C prior to hydroxyl radical measurement. Instrument settings are indicated in the figure legend. Signal intensity (peak height) was used to measure the relative amount of superoxide radicals produced and is measured in millimeters. All data are presented as peak height fold change above control animal AM response to Cr (VI).