Immune Cell Profiling of Prostate Tumor Microenvironment

Subcutaneous TRAMP tumors, prostate-containing tumor, and normal prostate tissue were minced into fragments, and incubated in collagenase solution in the presence of DNase I (1 mg/ml) at 37°C for 1 h. Dissociated cells were passed through a 100 µm cell strainer. Spleens or BM extracted from mouse femurs were homogenized and passed through a 100 µm cell strainer. Cells were resuspended and stained with direct labeled Abs against: CD45 (30-F11), CD11b (M1/70), Gr-1 (RB6-8C5), Ly6G (1A8) and Ly6C (HK1.4) (BioLegend); and B220 (RA3-6B2), F4/80 (BM8) and CD11c (N418) (eBioscience). For T-cell analysis, Abs from BioLegend were used: CD3 (145-2C11), CD4 (GK1.5), CD8 (53-6.7), IFNγ (XMG1.2) and Foxp3 (MF-14). Mouse G-CSF R/CD114 Ab was from R&D Systems. For intracellular staining, Abs against phospho-STAT3 (Y705) and phosphor-STAT5 (Y694) (BioLegend) were used. Staining was performed as previously described (25 (link), 31 (link)). All samples were analyzed on an LSRIIB FACS instrument and were further analyzed with FlowJo software.

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Yu H., Liu Y., McFarland B.C., Deshane J.S., Hurst D.R., Ponnazhagan S., Benveniste E.N, & Qin H. (2015). SOCS3 Deficiency in Myeloid Cells Promotes Tumor Development: Involvement of STAT3 Activation and Myeloid-Derived Suppressor Cells. Cancer immunology research, 3(7), 727-740.

Publication 2015

Ly6C (HK1.4) IFNγ (XMG1.2) Foxp3 (MF-14).

Cd11b Cells Collagenase Dnase i Femurs G csf r Ifnγ Intracellular Mouse Normal tissue Phosphor Prostate Prostate tumor Stat3 Stat5 T cell Tumors

Corresponding Organization : University of Alabama at Birmingham

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Variable analysis

independent variables

Subcutaneous TRAMP tumors
Prostate-containing tumor
Normal prostate tissue

dependent variables

Cell population analysis using flow cytometry for CD45, CD11b, Gr-1, Ly6G, Ly6C, B220, F4/80, CD11c, CD3, CD4, CD8, IFNγ, Foxp3, and G-CSF R/CD114
Intracellular staining for phospho-STAT3 (Y705) and phosphor-STAT5 (Y694)

control variables

Collagenase solution
DNase I (1 mg/ml)
Cell strainers (100 µm)
Antibodies used for staining
Staining protocol

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

Annotations

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