Pe cyanine5

Cells were washed once with phosphate-buffered saline (PBS) and then harvested with 0.05% trypsin/0.025% EDTA. Detached cells were washed with PBS containing 1% FCS and 1% penicillin/streptomycin (wash buffer), and resuspended in the wash buffer (10⁶ cells/100 μl). Combinations of fluorochrome-conjugated monoclonal antibodies obtained from BD Biosciences (San Diego, CA, USA) against human CD44 (PE-Cyanine5; cat. #15–0441) and CD24 (PE; cat. #15–0247) or their respective isotype controls were added to the cell suspension at concentrations recommended by the manufacturer and incubated at 4 °C in the dark for 30 to 40 min. The labeled cells were washed in the wash buffer, then fixed in PBS containing 1% paraformaldehyde, and then analyzed on BD FACSCalibur flow cytometer.

Single-cell suspensions were prepared from lymphocytes of thymus (T), bone marrow (BM), spleen (Spl), and lymph node (LN) from mice of the described genotypes and ~1 × 10⁵ cells in 1× PBS were stained for 15 min at RT using fluorescence-conjugated antibodies and analyzed by flow cytometry^{60 (link)}. The following antibodies were used: FITC-Cd11b (1:200, M1/70, BD Pharmingen, 553310), APC-Ly-6G/Ly-6C (Gr-1) (1:200, RB6-8C5, Biolegend), PE-IgM (1:400, SouthernBiotech, 1020-09), PE-CD4 (1:200, GK1.5, BD Pharmingen, 553730), FITC-CD8a (1:200, 53-6.7, Biolegend), PE-Cyanine5-CD3e (1:200, 145-2C11, Invitrogen, 15-0031-63).
For CSR assay, single-cell suspensions of spleen cells were sorted with CD43 magnetic beads (MACS, Miltenyi), and CD43- B cells were cultured at a density of 5 × 10⁵ cells per ml in RPMI medium supplemented with 15% FBS and 25 ng ml⁻¹ of IL-4 (R&D) and anti-CD40 (BD Biosciences). Cultured cells were maintained daily at a density of 1 × 10⁶ cells per ml. Cells were collected every day up to day 4.5 and were stained with FITC-conjugated IgG1 (1:200, A85-1, BD Pharmingen, 553443) and PE-Cyanine5-conjugated B220 (1:200, RA3-6B2, BD Pharmingen, 553091). Flow cytometry was performed on a FACSCalibur flow cytometer (BD Biosciences) and data were processed using FlowJo software package.