Kaluza 1.2 flow analysis software

The following monoclonal antibodies were used: anti-CD3-e450 (OKT3), anti-CD8a-APC-e780 (OKT8) (eBioscience, Vienna, Austria), anti-CD45RO-FITC (UCHL1), anti-CCR7-PE (3D12) (BD Bioscience, Breda, Netherlands), and anti-CD28 PeCy7 (CD28.2) (Biolegend, Uithoorn, The Netherlands). Cells were sorted using a MoFlo flow cytometry cell sorter (Backman Coulter, Woerden, The Netherlands).
Expression of cell surface markers on T cells was assessed using mAbs against human CD28-PE-CY7 (CD28.2) (Biolegend), CCR7-PE (3D12), and CD45RO-FITC (UCHL1) (BD Biosciences). Cells were analyzed using a BD LSR-II Flow Cytometer and the Diva software (BD Biosciences). Data analysis was done using the Kaluza Flow Analysis Software (1.2) (Beckman Coulter).

Cell surface markers on in vitro activated T cells were assessed using mAbs against human CD25-PE (BC96) (eBioscience, Vienna, Austria) and CD45RO-FITC (UCHL1) (BD Biosciences, Breda, The Netherlands). Cells were analyzed on BD LSR-II Flow Cytometer by Diva software (BD Biosciences). Data analysis was performed on Kaluza Flow Analysis Software (1.2) (Beckman Coulter, Woerden, The Netherlands).

Blood was collected in EDTA tubes, and 100 μl was incubated with anti-human CD19-eFluor450 (eBioscience, San Diego, CA, USA), CD24-fluorescein isothiocyanate (FITC) (BD biosciences, Franklin Lakes, NJ, USA), CD27-APC-eFluor780 (eBioscience), CD38-PeCy5 (eBioscience), CD5-PerCp-Cy5.5 (Biolegend, San Diego, CA, USA) or the corresponding isotype controls. After 15 minutes cells were treated with fluorescence-activated cell sorting (FACS) Lysing solution (BD Biosciences). Samples were measured using an LSR-II flow cytometer (BD biosciences) and data were analyzed using Kaluza 1.2 flow analysis software (Beckman Coulter, Brea, CA, USA). B cells were divided into transitional, memory, naive, CD24^highCD38^high and CD24^highCD27+ B cells as described previously [14 (link)]. CD5+ B cells were gated on an isotype control.