Quantifying Immune Cell Profiles in Intracranial Melanoma

Flow cytometry was used to analyze immune cell populations (12 (link), 13 (link), 44 (link)) in a minimum of three samples in two independent animal studies. Dissected intracranial melanoma tumors were physically dissociated and filtered through a 70-μm cell filter. Single cells were labeled with primary antibody. Cells without primary antibody labeling were used as unlabeled negative controls; fluorescent beads (UltraComp eBeads^™, Invitrogen^™, Carlsbad, CA) were used as positive/calibration controls and to determine compensation between fluorescent channels. Forward- and side-scatter gating identified single cells and viable cell (Ghost Dye^™ Red 780 Viability Dye, 1:100; Tonbo Biosciences, San Diego, CA) exclusion identified live cells. Fluorescence minus one (FMO) methodology was used to determine gating. Flow cytometry was performed on an Attune NxT Flow Cytometer (Thermo Fisher Scientific^™ Inc., Waltham, MA), and compensation matrix computed and data analyzed using FlowJo version 9 software (Ashland, OR) following published flow cytometry guidelines (45 (link)).
Antibodies used were: CD4 (BV510, 1:400, clone RM4–5), CD8 (PerCP-cy5.5, 1:200, clone 53–6.7), FOXP3 (BV421, 1:100, clone MF-14), all acquired from BioLegend^® Inc. (San Diego, CA); and CD45 (PE-cy7, 1:200, clone 30-F11), CD3 (FITC, 1:200, clone 17A2), both acquired from Tonbo Biosciences.

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Clark P.A., Sriramaneni R.N., Bates A.M., Jin W.J., Jagodinsky J.C., Hernandez R., Le T., Jeffery J.J., Marsh I.R., Grudzinski J.J., Aluicio-Sarduy E., Barnhart T.E., Anderson B.R., Chakravarty I., Arthur I.S., Kim K., Engle J.W., Bednarz B.P., Weichert J.P, & Morris Z.S. (2021). Low-Dose Radiation Potentiates the Propagation of Anti-Tumor Immunity against Melanoma Tumor in the Brain after In Situ Vaccination at a Tumor outside the Brain. Radiation research, 195(6), 522-540.

Publication 2021

UltraComp eBeads Ghost Dye™ Red 780 Viability Dye, Attune NxT Flow Cytometer CD4 (BV510, ), CD8 (PerCP-cy5.5, FOXP3 (BV421, CD45 (PE-cy7,

Animal Antibodies Antibody Cells Clone Cy5 5 Fitc Flow cytometry Fluorescence Ghost Intracranial tumors Melanoma Populations

Corresponding Organization :

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

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Variable analysis

independent variables

Animal studies
Intracranial melanoma tumors

dependent variables

Immune cell populations

control variables

Minimum of three samples
Two independent animal studies
Dissected intracranial melanoma tumors physically dissociated and filtered through a 70-μm cell filter
Single cells labeled with primary antibody
Cells without primary antibody labeling used as unlabeled negative controls
Fluorescent beads (UltraComp eBeads™, Invitrogen™, Carlsbad, CA) used as positive/calibration controls and to determine compensation between fluorescent channels
Forward- and side-scatter gating identified single cells and viable cell (Ghost Dye™ Red 780 Viability Dye, 1:100; Tonbo Biosciences, San Diego, CA) exclusion identified live cells
Fluorescence minus one (FMO) methodology used to determine gating
Flow cytometry performed on an Attune NxT Flow Cytometer (Thermo Fisher Scientific™ Inc., Waltham, MA), and compensation matrix computed and data analyzed using FlowJo version 9 software (Ashland, OR) following published flow cytometry guidelines (45 (link))
Antibodies used: CD4 (BV510, 1:400, clone RM4–5), CD8 (PerCP-cy5.5, 1:200, clone 53–6.7), FOXP3 (BV421, 1:100, clone MF-14), all acquired from BioLegend® Inc. (San Diego, CA); and CD45 (PE-cy7, 1:200, clone 30-F11), CD3 (FITC, 1:200, clone 17A2), both acquired from Tonbo Biosciences

positive controls

Fluorescent beads (UltraComp eBeads™, Invitrogen™, Carlsbad, CA) used as positive/calibration controls and to determine compensation between fluorescent channels

negative controls

Cells without primary antibody labeling used as unlabeled negative controls

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