Multiparametric Flow Cytometry of Tumor Cells

Portions of primary tumor and metastatic tissues were digested with collagenase IV/DNase (0.2 mg/mL and 10 μg/mL) at 37 °C for 30 min. Cells were put through 40-μm filters to obtain single-cell suspensions and stained as previously described [21 (link)]. Cell surfaces were stained with anti-mouse CD4, anti-mouse CD206, anti-mouse CD11b, anti-mouse F4/80, anti-mouse Gr1.1, anti-mouse CD3, anti-mouse CD45, anti-mouse CD8a, anti-mouse CD28, anti-mouse NKG2D, anti-human CD45, anti-human CD8, anti-human CD33, and anti-human HLA-DR antibodies (BioLegend, San Diego, CA) labeled with fluorescein isothiocyanate, PerCP/Cy5.5, phycoerythrin, or Brilliant Violet 421. Flow cytometry was performed using an Attune flow cytometer (Life Technologies), and the data were analyzed with FlowJo software.

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Zhao Q., Hu J., Mitra A., Cutrera J., Zhang W., Zhang Z., Yan J., Xia X., Mahadeo K.M., Livingston J.A., Gorlick R, & Li S. (2019). Tumor-targeted IL-12 combined with tumor resection yields a survival-favorable immune profile. Journal for Immunotherapy of Cancer, 7, 154.

Publication 2019

anti-mouse CD4 anti-mouse CD206 anti-mouse F4/80 anti-mouse CD3 anti-mouse CD45 anti-mouse CD28 anti-human CD45 anti-human CD8 anti-human CD33 anti-human HLA-DR Attune flow cytometer

Anti antibodies Anti cd3 Cd11b Cell Collagenase Cy5 5 Dnase iv Flow cytometry Fluorescein Hla dr Human Isothiocyanate Mouse Phycoerythrin Tissues Tumor Violet

Corresponding Organization :

Other organizations : The University of Texas MD Anderson Cancer Center, Pfizer (United States)

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Variable analysis

independent variables

Collagenase IV/DNase (0.2 mg/mL and 10 μg/mL)

dependent variables

Cell suspension obtained after filtration through 40-μm filters
Cell surface expression of CD4, CD206, CD11b, F4/80, Gr1.1, CD3, CD45, CD8a, CD28, NKG2D, CD45, CD8, CD33, and HLA-DR

control variables

Cell staining procedure as previously described [21]
Flow cytometry using an Attune flow cytometer (Life Technologies)

controls

No positive or negative controls were explicitly mentioned.

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