Igm alexa fluor 647

To evaluate the proliferation and differentiation of B cells after stimulation with CpG, cells were stained with the appropriate combination of fluorochrome-conjugated Abs to identify B cell subsets: CD19 BB700 (clone SJ25C1 Becton Dickinson), CD27 PE (clone M-T271 Becton Dickinson), CD38 BV421 (clone HIT2, Becton Dickinson), and IgM Alexafluor647 (conjugated Affinipure F(ab’)2, Jackson ImmunoResearch Laboratories, West Grove, PA, USA). Dead cells were excluded from analysis by side/forward scatter gating. All analyses were performed on a LSRFortessaX-20 (Becton Dickinson, San Jose, CA, USA) interfaced to a FACSDiva software (BD Biosciences, San Jose, CA, USA). Fifty thousand gated events on living cells were analyzed, whenever possible, for each sample.

After 4- and 7-days of CpG stimulation, PBMC were harvested from the culture plates and washed by centrifugation at 1200 rpm for 5 minutes at room temperature (RT) in wash buffer (2% FBS in PBS). To identify B cell subsets, single-cell suspensions were stained on day 0 and after 4–7 days of stimulation with CpG or incubation with IL-2 alone with the appropriate combination of the following directly conjugated monoclonal antibodies (MoAb): CD19-Alexa Fluor 700 (1∶90 dilution; BD Biosciences, San Diego, CA, USA), CD38-PECy7 (1∶90 dilution; BD Biosciences), CD24-FITC (1∶10 dilution, BD Biosciences), CD27-PE (1∶20 dilution; BD Biosciences) and IgM-Alexa Fluor 647 (1∶400 dilution; Jackson ImmunoResearch, West Baltimore, Pike, PA, USA). The cells were incubated for 20 minutes in the dark at 4°C. After labeling, the cells were washed by centrifugation at 1200 rpm for 5 minutes at 4°C in wash buffer. Data were acquired on a FACSCanto II. Flow cytometry profiles were analyzed using FACSDiva software (BD Biosciences, San Jose, CA, USA). Dead cells were excluded from the analysis by side/forward scatter gating. A minimum of fifty thousand gated events on living cells were collected per data set.