Molecular probes countbright beads

After FACS sorting, cells were labeled with Efluor670 or CFSE at previously determined optimal concentrations, by incubation at 37°C for 10 mins., then washed three times with staining medium containing 10% neonatal calf serum and resuspended into ‘Culture Media’ (RPMI 1640 with 10% heat inactivated fetal bovine serum, 292 µg/ml L-glutamine, 100 Units/ml penicillin, 100 µg/ml streptomycin, and 50 µM 2-mercaptoethanol). Cells were plated at 10⁵ cells/well of 96-well U bottom tissue culture plates (BD Bioscience), and unless otherwise indicated, cultured at 37°C/5% CO₂ for 3 days. When indicated, LPS at 10 µg/ml, Mycobacterium TB lipids at 20 µg/ml (BIA), Imiquimod (R837, InvivoGen) at 1 µg/ml, CpG ODN 7909 at 5 µg/ml or anti-IgM (Fab)₂ at 10–20 ug/ml were added to the wells. Cell enumeration after culture was performed using Molecular Probes CountBright Beads (Thermo Fisher) by flow cytometry, per manufacturer instructions. After culture, culture plates were spun and supernatant was collected and stored at −20°C, and cells were stained for FACS.

Fluorescently labeled antibodies specific for CD3 (Alexa Fluor 700; Life Technologies, Carlsbad, CA, USA), CD4 (PE or Pacific Blue), CD8 (allophycocyanin [APC]), CD19 (peridinin chlorophyll-cyanine 5.5 [PerCP-Cy5.5]), CD25 (PerCP-Cy7), and CD62L (fluorescein isothiocyanate [FITC]) were obtained from BD Biosciences (San Jose, CA, USA). Intracellular staining for Foxp3 was performed using anti-mouse Foxp3 antibody labeled with FITC according to the manufacturer’s instructions (eBioscience, San Diego, CA, USA). Labeled cells were washed with phosphate-buffered saline (PBS), and a minimum of 10,000 cells were analyzed from each sample using a 5-laser (355, 405, 488, 561, and 640 nm) BD LSR II flow cytometer (BD Biosciences). To determine cell numbers, 50 μl of Molecular Probes CountBright beads (Thermo Fisher Scientific, Eugene, OR, USA) were added to each sample before cytometric analyses, and the number of cells per unit volume was calculated as (cell events/bead events) × (bead count/volume of blood sample analyzed). Data analysis was performed using FlowJo software (Tree Star, Ashland, OR, USA).