Igg1 fitc

Phenotyping of cell surface markers was performed by flow cytometry. The cells were stained with CD34-PE and CD45-FITC (all from BD Biosciences, USA), CD90-FITC (Dako, USA), CD73 PE (Abcam, USA) and isotype controls IgG1-FITC (Dako, USA), IgG1-PE (BD Biosciences, USA), and IgG2A-APC (BD Biosciences, USA). Flow cytometry data were acquired using a Guava EasyCyte 8HT flow cytometer and analysed using ExpressPro software (Merck Millipore, USA) comparing unlabelled, marker-labelled and isotype control populations in FL-1, FL-2 and FL-4 channels.

CBMCs were analysed by flow cytometry (BD Biosciences, San Jose, CA, USA) after three days of cultivation. For surface staining, 2 μl of anti-human CD4-fluorescein isothiocyanate (FITC), 1 μl of CD25-RPE-Cy5, 1 μl of IgG1-FITC (DakoCytomation, Glostrup, Denmark), and 0.5 μl of IgG2a RPE-Cy5 (BD Biosciences) were added. For CD4/CD25/FOXP3 co-staining, 8 μl of anti-human CD4-FITC and 4 μl of anti-human CD25-RPE-Cy5 antibodies were added to 1 × 10⁶ cells in 100 ml PBS. Cells were then permeabilized and FOXP3-PE or the corresponding isotype control antibodies were added. Data were analysed with CellQuest software (BD Biosciences).

For staining of peripheral blood mononuclear cells with purified antibodies, 100,000 cells were incubated with 0.2μg of antibody diluted in PBS/0.5% BSA/0.02% NaN3 (FACS medium) for 1h on ice as previously described [42 (link), 43 (link)]. Cells were centrifuged 400g/5min and supernatant was removed. The cells were incubated 30mins on ice with secondary antibodies, washed with FACS medium and analyzed with FACS calibur instrument (BD, Franklin Lakes, NJ, USA) and FlowJo or CellQuest software. The following antibodies were used: PE-anti-CD56 (DAKO, Glostrup, Denmark, clone MOC-1), FITC-anti CD3 (DAKO, clone UCHT1), APC-anti-NKG2D (eBioscience, San Diego, USA, clone CX5), APC-anti-NKp46 (eBioscience). Isotype control IgG1-FITC (DAKO), IgG1-PE (DAKO) and IgG1-APC (eBioscience) served as negative controls. NK cells were gated based on positive CD56 expression and lack of CD3 expression. At least 2,500 gated NK cells were analyzed in each reading.