Purified Mouse Anti-Human CD135

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	Cells were incubated with antibodies at 4 °C for 30 min.

Protocol tips
Cells were incubated with antibodies at 4 °C for 30 min.

Publication protocol

AML blasts from peripheral blood cells or bone marrow cells from untreated patients with AML were studied after informed consent was obtained in accordance with the Declaration of Helsinki, and the protocol was approved by the Medical Ethical Committee. AML mononuclear cells were isolated by density gradient centrifugation, and CD34+ cells were stained using CD34-PE antibody (BD Biosciences, San Jose, CA, USA) and selected by sorting on a MoFLo (DakoCytomation, Carpinteria, CA, USA). AML cocultures were performed on MS5 stromal cells as described previously (19, 20). All fluorescence-activated cell sorter (FACS) analyses were performed on a FACScalibur (Becton-Dickinson [BD], Alpen a/d Rijn, the Netherlands) and the data were analyzed using WinList 3D (Verity Software House, Topsham, USA) or FlowJo (Tree Star, Oregon, USA) software. Cells were incubated with antibodies at 4 °C for 30 min. Antibodies against CD34, CD38, CD135, CD47, and ITGA6 were obtained from BD Biosciences (Breda, The Netherlands), antibodies against CD96 and PTH2R were obtained from Santa Cruz (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and second goat-anti-rabbit-FITC antibodies (used for PTH2R stains) were obtained from Invitrogen (Breda, The Netherlands).

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