Isolation and Characterization of MCL Subpopulations

MCL tumor cell-enriched buffy coats were isolated from apheresis or leukemic phase blood of MCL patients by Histopaque-1077 (Sigma-Aldrich, St. Louis, MO) gradient centrifugation. Obtained cells were then stained with antibodies against CD34-APC (Cat No. 555824), CD3-APC-Cy7 (Cat No. 557832), CD45-FITC (Cat No. 555482), CD19-PE (Cat No. 555413), and Sytox blue for selection of live cells (all from BD Bioscience, San Jose, CA). Subpopulations of MCL-ICs (CD34-CD3-CD45+CD19-) and MCL-non-ICs (CD34-CD3-CD45+CD19+) were isolated using a fluorescence-activated cell sorter (Influx, BD Bioscience, San Jose, CA) according to a previously described protocol [25 (link)]. Subpopulations of sorted cells were analyzed for purity by immunostaining with markers for plasma cells (CD27, CD38) and natural killer cells (CD56, CD16) using the antibodies CD27-PerCP-Cy5.5 (Cat No. 560612), CD38-PE-Cy7 (Cat No. 560677), CD56-PE-Cy7 (Cat No. 557747), and CD16-Pacific blue (Cat No. 558122) (all from BD Bioscience, San Jose, CA), respectively.

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Mathur R., Sehgal L., Braun F.K., Berkova Z., Romaguerra J., Wang M., Rodriguez M.A., Fayad L., Neelapu S.S, & Samaniego F. (2015). Targeting Wnt pathway in mantle cell lymphoma-initiating cells. Journal of Hematology & Oncology, 8, 63.

Publication 2015

Histopaque-1077 CD34-APC CD3-APC-Cy7 CD45-FITC CD19-PE Sytox blue CD27-PerCP-Cy5.5 CD38-PE-Cy7 CD56-PE-Cy7 CD16-Pacific blue

Antibodies Apheresis Blood Cell coats Cells Centrifugation Cy5 5 Fitc Fluorescence Histopaque Natural killer cells Patients Plasma cells Subpopulations Tumor

Corresponding Organization :

Other organizations : The University of Texas MD Anderson Cancer Center

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Variable analysis

independent variables

Isolation of MCL tumor cell-enriched buffy coats from apheresis or leukemic phase blood of MCL patients by Histopaque-1077 gradient centrifugation
Staining of obtained cells with antibodies against CD34-APC, CD3-APC-Cy7, CD45-FITC, CD19-PE, and Sytox blue

dependent variables

Subpopulations of MCL-ICs (CD34-CD3-CD45+CD19-) and MCL-non-ICs (CD34-CD3-CD45+CD19+) isolated using a fluorescence-activated cell sorter
Purity analysis of sorted cell subpopulations by immunostaining with markers for plasma cells (CD27, CD38) and natural killer cells (CD56, CD16)

control variables

Not explicitly mentioned

controls

No positive or negative controls were explicitly mentioned in the provided information.

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