CMML Patient Sample Collection and Cell Line Processing

Chronic myelomonocytic leukemia patient samples were collected at the Division of Hematology, Medical University of Graz, Austria, as well as in the Austrian Biodatabase for CMML. All samples were processed and stored as described in detail in the Online Supplementary Methods. Healthy CD34⁺ HSPC were collected from umbilical cord blood specimens (EasySep, STEMCELL Technologies) according to the manufacturerś instructions and processed as described before.^{24 (link)} Peripheral blood samples from healthy donors were used to collect CD14⁺ monocytes (MACS, Miltenyi Biotec), B lymphocytes and granulocytes (LymphoprepTM, STEMCELL Technologies and human B Lymphocyte enrichment set, BD biosciences) according to the manufacturer’s protocol. 293T, NB4 and HL-60 cell lines were obtained from the German National Resource Center for Biological Material (DSMZ, Braunschweig, Germany). Low passage stocks were frozen and cells were always passaged for less than six months after resuscitation. Additionally, cells were screened by variable number of tandem repeat profiling (VNTR) for authenticity.^{21 (link)} Lentiviral transduction of cell lines and primary HSPC were performed as previously described.^{12 (link),21 (link),22 (link)}

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Caraffini V., Geiger O., Rosenberger A., Hatzl S., Perfler B., Berg J.L., Lim C., Strobl H., Kashofer K., Schauer S., Beham-Schmid C., Hoefler G., Geissler K., Quehenberger F., Kolch W., Athineos D., Blyth K., Wölfler A., Sill H, & Zebisch A. (2020). Loss of RAF kinase inhibitor protein is involved in myelomonocytic differentiation and aggravates RAS-driven myeloid leukemogenesis. Haematologica, 105(2), 375-386.

Publication 2019

EasySep MACS LymphoprepTM

B lymphocyte Biological Cell lines Cells Cmml Frozen Granulocytes Human Monocytes Patient Peripheral blood donors Resuscitation Stemcell Umbilical cord blood Variable number of tandem repeat

Corresponding Organization :

Other organizations : Medical University of Graz, Klinik Hietzing, Sigmund Freud University Vienna, University College Dublin, Cancer Research UK Scotland Institute

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Protocol cited in 4 other protocols

Variable analysis

independent variables

Lentiviral transduction of cell lines and primary HSPC

dependent variables

Not explicitly mentioned

control variables

Healthy CD34+ HSPC collected from umbilical cord blood specimens
Peripheral blood samples from healthy donors used to collect CD14+ monocytes, B lymphocytes, and granulocytes
293T, NB4 and HL-60 cell lines obtained from the German National Resource Center for Biological Material (DSMZ, Braunschweig, Germany)
Low passage stocks of cell lines were frozen and cells were always passaged for less than six months after resuscitation
Cells were screened by variable number of tandem repeat profiling (VNTR) for authenticity

positive controls

Healthy CD34+ HSPC collected from umbilical cord blood specimens
Peripheral blood samples from healthy donors used to collect CD14+ monocytes, B lymphocytes, and granulocytes

negative controls

293T, NB4 and HL-60 cell lines obtained from the German National Resource Center for Biological Material (DSMZ, Braunschweig, Germany)
Low passage stocks of cell lines were frozen and cells were always passaged for less than six months after resuscitation
Cells were screened by variable number of tandem repeat profiling (VNTR) for authenticity

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