Mouse isotype controls

Monoclonal antibodies recognizing the following surface antigens were used: pan HLA class II (6604366, 1:500, Beckman Coulter), HLA-DP (ab21119-100, 1:100, Abcam), HLA-DR (555561, 1:500, BD Biosciences), Ii (555540, 1:500, BD Biosciences), CLIP (555981, 1:200, BD Biosciences), HLA-DM (555983, 1:250, BD Biosciences), NGFR (557196, 1:200, BD Biosciences). Mouse isotype controls were from BD Biosciences and each was used at 1:500. Surface and intracellular molecular staining was carried out as described elsewhere21 (link)49 (link).

Flow cytometry (FCM) was used to detect clusters of immunocytes in patients with MDS. Fresh whole blood samples collected into EDTA (100 μL) were immunostained with anti-CD3-PerCP, anti-CD8-FITC, anti-CD4-PE, anti-CD56-APC, anti-CD16-FITC, anti-CD19-APC, and mouse isotype controls (BD Biosciences, San Jose, CA, USA) in separate Trucount tubes (BD Biosciences, USA) for 30 min at 4°C in the dark, followed by erythrocyte lysis using 2 mL erythrocyte lytic solution (BD PharMingen, San Diego, CA, USA). After centrifugation at 1300 rpm for 5 min, the supernatant was discarded. Cells were washed with phosphate-buffered saline (PBS) and resuspended in 300 μL PBS, and at least 50,000 cells were acquired on a FACSCalibur flow cytometer (BD Biosciences, USA). Analysis was done using CellQuest software version 3.1 (Becton Dickinson, Franklin Lakes, NJ, USA).

For analysis of apoptosis by flow cytometry, propidium iodide (PI) and FITC conjugated annexin-V (IQ products, Netherlands) were used with the appropriate mouse isotype controls (BD PharMingen, San Diego, CA). All antibodies were used at concentrations titrated for optimal staining and carried out using standardized protocols.