Multicolor Flow Cytometry Analysis

Single-cell suspensions from spleens, dLNs, non-dLNs, or tumor tissues were prepared as previously described (42 (link)). Combinations of the following fluorochrome-conjugated antibody were used for cell surface or intracellular staining to define populations of NK, CD8, and subsets of CD4 T cells: CD3e (clone 145-2c11), CD8a (clone 53-6.7), CD4 (clone GK1.5), NK1.1 (clone PK136), NKG2D (clone CX5), CD44 (clone eBio4B10), CD11c (clone N418), MHCII (clone M5/114.15.2), CD80 (clone 16-10A1), CD86 (clone PO3), CD40 (clone 1C10), and CD3ζ (clone 6B10.2, BioLegend). For ex vivo restimulation, freshly isolated single-cell suspension was cultured in complete RPMI 1640 medium containing PMA (50 ng/ml) and ionomycin (500 ng/ml) for 6 hours before it was analyzed for IFN-γ production by intracellular staining with an antibody specific to IFN-γ (XMG1.2). Multicolored flow cytometry analyses were performed on LSR II (BD). Data were analyzed with FlowJo software (Tree Star).

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Zhang J., Liu D., Li G., Staveley-O’Carroll K.F., Graff J.N., Li Z, & Wu J.D. (2017). Antibody-mediated neutralization of soluble MIC significantly enhances CTLA4 blockade therapy. Science Advances, 3(5), e1602133.

Publication 2017

CD3e (clone 145-2c11), CD8a (clone 53-6.7), CD4 (clone GK1.5), CD11c (clone N418), MHCII (clone M5/114.15.2), CD80 (clone 16-10A1),

Antibody Antibody specific Cd44 Cell Clone Cultured medium Flow cytometry Fluorochrome Ifn γ Intracellular Ionomycin Populations Subsets t cells Tissues Tree Tumor

Corresponding Organization :

Other organizations : Medical University of South Carolina, University of Missouri, Oregon Health & Science University, MUSC Hollings Cancer Center

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Variable analysis

independent variables

Combinations of the following fluorochrome-conjugated antibody used for cell surface or intracellular staining: CD3e, CD8a, CD4, NK1.1, NKG2D, CD44, CD11c, MHCII, CD80, CD86, CD40, and CD3ζ
PMA (50 ng/ml) and ionomycin (500 ng/ml) used for ex vivo restimulation

dependent variables

Populations of NK, CD8, and subsets of CD4 T cells defined by the cell surface or intracellular staining
IFN-γ production measured by intracellular staining

control variables

Single-cell suspensions from spleens, dLNs, non-dLNs, or tumor tissues prepared as previously described

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

Annotations

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