PE anti-human CD52 Antibody

Protocol tips

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	Heparinized bone marrow (BM) and peripheral blood (PB) cells (106 leukocytes per tube) were incubated with various combinations of mAb (Supplementary Table S3) for 15 minutes. Then, erythrocytes were lysed in FACS-Lysing-Solution (BD Biosciences, San José, CA, USA). Washed cells were acquired on a FACSCalibur (BD Biosciences) and analyzed by FlowJo software (TreeStar, Ashland, OR, USA) as reported

Protocol tips

Heparinized bone marrow (BM) and peripheral blood (PB) cells (106 leukocytes per tube) were incubated with various combinations of mAb (Supplementary Table S3) for 15 minutes. Then, erythrocytes were lysed in FACS-Lysing-Solution (BD Biosciences, San José, CA, USA). Washed cells were acquired on a FACSCalibur (BD Biosciences) and analyzed by FlowJo software (TreeStar, Ashland, OR, USA) as reported

Publication protocol

Heparinized bone marrow (BM) and peripheral blood (PB) cells (106 leukocytes per tube) were incubated with various combinations of mAb (Supplementary Table S3) for 15 minutes. Then, erythrocytes were lysed in FACS-Lysing-Solution (BD Biosciences, San José, CA, USA). Washed cells were acquired on a FACSCalibur (BD Biosciences) and analyzed by FlowJo software (TreeStar, Ashland, OR, USA) as reported (Blatt et al., 2014; Herrmann et al., 2012, 2014). Antibody-reactivity was controlled using isotype-matched control-antibodies. The gating-strategy to detect CD34+/CD38− stem cells and CD34+/CD38+ progenitor cells is shown in Supplementary Figure S1. Flow cytometric results were expressed as ratio of median fluorescence intensities (MFI) obtained with specific mAb and control mAb (MFI mAb: MFI control mAb). The MFI ratio was graded according to a published score (Blatt et al., 2014; Herrmann et al., 2012, 2014): MFI ratio >100: +++, MFI ratio 10.01-100: ++, MFI ratio 3.01-10: +, MFI ratio 1.51-3: +/−, MFI ratio ≤1.50: –. In a separate set of experiments, cell lines were incubated with imatinib, nilotinib, ponatinib, BEZ235, RAD001, RDEA119, PD0325901 (each 0.1-1 µM), or pimozide (1-10 µM) at 37°C for 24 hours before stained with a PE-conjugated CD25 mAb or IL-1RAP mAb.

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