From our previous study (Pokkali et al., 2009), an MOI of 3 was found optimum for infecting PMNs, and hence, same was kept as standard throughout this study. Because we aimed at observing the initial effect of mycobacterial vaccine strains on neutrophils, early time point
of 4 h was chosen. Uninfected neutrophils (Control) served as negative BAY 80-6946 mouse control, and 10 nm phorbol myristate acetate (PMA) (Sigma Chemicals)–stimulated cells were used as positive control. After 4 h, the neutrophil culture supernatants (Nu sups) were collected, centrifuged, and used to stimulate peripheral blood mononuclear cells (PBMCs), and the remaining was stored in aliquots at −70 °C until use. The cells were washed with PBS twice and used for fluorescence-activated cell sorting (FACS) staining protocol as given in the section ‘cell phenotyping https://www.selleckchem.com/products/BAY-73-4506.html by flow cytometry’. The buffy coat containing PBMCs was collected after Ficoll-Hypaque density gradient centrifugation. The cells were washed once with Hanks’ balanced salt solution (HBSS) and suspended in RPMI 1640 medium supplemented with 1% FBS. The cell viability was always found to be > 95% through trypan
blue exclusion test, and the cell density was adjusted to 1 × 106 mL−1. The cells were stimulated with 200 μL of infected Nu sups and cultured in 12 Well Clear TC-Treated Multiple Well Plates (Corning
Life Sciences) for 18 h at 37 °C in a humidified 5% CO2 incubator. After 18 h, the cells were harvested and stained for FACS as given in the section ‘cell phenotyping by flow cytometry’. Cell Fluorouracil manufacturer surface expression of CD32, CD64, TLR-4, and CXCR3 on neutrophils (CD16+ve); CD69 and CXCR3 on T helper cells (CD4+ve); and CCR5 and CCR7 on monocytes (CD14+ve) was determined by staining the cells using the monoclonal mouse anti-human conjugated antibodies, i.e. CD16 (clone 3G8)–fluorescein isothiocyanate (FITC), TLR-4 (clone HTA125)–phycoerythrin (PE), CD32 (clone FL18.26), CD64 (clone 10.1), CD4 (clone RPA T4), CD14 (clone M5E2)–allophycocyanin (APC), CD69 (clone FN50)–phycoerythrin-cyanine5 (PE-Cy5) (BD Pharmingen), and CCR5 (clone 45549)–FITC, CCR7 (clone 150503), CXCR3 (clone 49801)–PE (R & D Systems), and their fluorescence emission was detected in FL-1 (FITC), FL-2 (PE), FL-3 (PE-Cy5), and FL-4 (APC) channels. The above specified clones were used throughout the study. Briefly, cells were incubated with PBS containing the combinations of antibodies at saturation for 20 min at 4 °C. Cells were washed and fixed with 1% paraformaldehyde (Sigma Chemicals) in PBS and analyzed on a FACSCalibur flow cytometer (Becton Dickinson).