Protocol detail

Multiparametric Flow Cytometry Analysis

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Single cell suspensions of bone marrow (BM) cells and splenocytes were prepared as described previously50 (link). All staining was performed in Dulbecco’s phosphate-buffered saline (DPBS; Gibco Life Technologies) supplemented with 2% fetal bovine serum (FBS; Sigma) on ice. Cells were blocked using FcR blocking reagent (Miltenyi Biotech, 1:20) and stained with combinations of the antibodies listed in Table S27. Lymphoid progenitors were analysed using APC-mouse lineage antibody cocktail (558074, BD Biosciences), FITC anti-CD127 (IL7-Ra), PE-Cy7 anti-c-Kit and PB anti- Sca1 (Table S27). Dead cells were excluded by gating on 7AAD (420403, Biolegend) negative cells. Fluorescence-minus-one controls were used to set appropriate gates. Flow cytometry analysis were performed on a CyAn ADP flow cytometer (Dako) or an LSR Fortessa cell analyser (BD Biosciences) and data were analysed with FlowJo software v 10 (Tree Star).

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Horton S.J., Giotopoulos G., Yun H., Vohra S., Sheppard O., Bashford-Rogers R., Rashid M., Clipson A., Chan W.I., Sasca D., Yiangou L., Osaki H., Basheer F., Gallipoli P., Burrows N., Erdem A., Sybirna A., Foerster S., Zhao W., Sustic T., Harrison A.P., Laurenti E., Okosun J., Hodson D., Wright P., Smith K.G., Maxwell P., Fitzgibbon J., Du M.Q., Adams D.J, & Huntly B.J. (2017). Early loss of Crebbp confers malignant stem cell properties on lymphoid progenitors. Nature cell biology, 19(9), 1093-1104.

Publication 2017

Dulbecco’s phosphate-buffered saline fetal bovine serum (FBS FcR blocking reagent APC-mouse lineage antibody cocktail FITC anti-CD127 7AAD CyAn ADP flow cytometer LSR Fortessa cell analyser

7aad Antibodies Antibody cocktail Bone marrow cells Cell Fitc Flow cytometry Fluorescence Lymphoid Mouse Phosphate Saline Sca1 Tree

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Variable analysis

independent variables

Cell type (bone marrow cells, splenocytes)

dependent variables

Lymphoid progenitor populations

control variables

Antibodies used for staining
Blocking with FcR blocking reagent
Gating on 7AAD-negative cells to exclude dead cells
Fluorescence-minus-one controls to set appropriate gates

controls

Positive controls: Not explicitly mentioned.
Negative controls: Fluorescence-minus-one controls used to set appropriate gates.

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