Flow Cytometric Analysis of Tumor Immune Cells

Portions of some tumors were processed for flow cytometry as previously described [31 (link)]. Single cell suspensions were stained with surface antibodies (Additional file 1: Table S2) and then fixed using the eBioscience FOXP3 fixation/permeabilization kit. UltraComp Beads eBeads (Invitrogen) were used for compensation and fluorescence minus one (FMO) methodology to determine gating. Flow cytometry was performed on an Attune Nxt flow cytometer (ThermoFisher), and compensation matrix and data were analyzed using FlowJo software. Tumor infiltrating innate and adaptive immune cell subpopulations were quantitated using the gating strategy shown in Additional file 2: Fig. S2A and B, respectively.

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O’Leary K.A., Bates A.M., Jin W.J., Burkel B.M., Sriramaneni R.N., Emma S.E., Nystuen E.J., Sumiec E.G., Ponik S.M., Morris Z.S, & Schuler L.A. (2023). Estrogen receptor blockade and radiation therapy cooperate to enhance the response of immunologically cold ER+ breast cancer to immunotherapy. Breast Cancer Research : BCR, 25, 68.

Publication 2023

FOXP3 fixation/permeabilization kit UltraComp Beads eBeads Attune Nxt flow cytometer

Adaptive Cell Flow cytometry Fluorescence Subpopulations Surface antibodies Tumor

Corresponding Organization :

Other organizations : University of Wisconsin–Madison, University of Wisconsin Carbone Cancer Center

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Tumor infiltrating innate and adaptive immune cell subpopulations

control variables

None explicitly mentioned

controls

UltraComp Beads eBeads (Invitrogen) were used for compensation and fluorescence minus one (FMO) methodology to determine gating.

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