Comprehensive Immune Profiling of Tumor Microenvironment

MC38, YUMM2.1, and YUMM1.1 tumors and spleens were harvested from mice at pre-defined time points. Tumors were digested with collagenase D (Roche), and stained with antibodies to CD3 BV605, Ly6C FITC, PD-L1/CD274 PE, CD8a BV421, CD45RA/B220, CD11b BV785, CD11c PECy7, CD103 PerCP Cyanine 5.5, MHC Class II (I-A/I-E) FITC (Biolegend, San Diego, CA), Ly6G (Gr-1) PerCP Cyanine 5.5, F4/80 Pacific blue/eFluor450, CD25 APC, CD4 FITC (eBioscience, San Diego, CA). Intracellular staining of Foxp3 PE (eBioscience) was done according to manufacturer's recommendations. Cells were analyzed with a LSR-II or FACSCalibur flow cytometer (BD Biosciences, San Jose, CA), followed by Flow-Jo software (Tree-Star, Ashland, OR) analysis (28 (link)).

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Moreno B.H., Zaretsky J.M., Garcia-Diaz A., Tsoi J., Parisi G., Robert L., Meeth K., Ndoye A., Bosenberg M., Weeraratna A.T., Graeber T.G., Comin-Anduix B., Hu-Lieskovan S, & Ribas A. (2016). Response to programmed cell death-1 blockade in a murine melanoma syngeneic model requires costimulation, CD4, and CD8 T cells. Cancer immunology research, 4(10), 845-857.

Publication 2016

collagenase D CD3 BV605 Ly6C FITC PD-L1/CD274 PE CD11b BV785 CD11c PECy7 CD25 APC CD4 FITC Foxp3 PE FACSCalibur flow cytometer

Antibodies Cd103 Cd11b Cd25 Cd274 Cells Collagenase Fitc Intracellular Mhc class ii Mice Tree Tumors

Corresponding Organization : Comprehensive Blood & Cancer Center

Other organizations : Yale University, The Wistar Institute, Howard Hughes Medical Institute

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Variable analysis

independent variables

Tumor type (MC38, YUMM2.1, YUMM1.1)

dependent variables

Immune cell populations (CD3, Ly6C, PD-L1/CD274, CD8a, CD45RA/B220, CD11b, CD11c, CD103, MHC Class II, Ly6G, F4/80, CD25, CD4, Foxp3)

control variables

Collagenase D (Roche) used for tumor digestion
Antibodies used for staining
Flow cytometry analysis with LSR-II or FACSCalibur flow cytometer (BD Biosciences)
Flow-Jo software (Tree-Star, Ashland, OR) used for data analysis

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

Annotations

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