Immune Cell Profiling of GD2-Expressing Tumors

9464D-GD2 tumors were extracted on day 13 and incubated for 30 min at 37 °C in dissociation solution containing HBSS supplemented with 5% FBS, 1 mg/mL collagenase type D, and 100 μg/mL DNase I (Sigma-Aldrich) as previously described [12 (link)]. For cell surface staining, cells were incubated with anti-GD2-APC (clone 14G2a; BioLegend), anti-CD45-eF450 (clone 30-F11; eBioscience), anti-CD3-Alexa700 (clone 17A2; BioLegend), anti-CD4-PE-Dazzle594 (clone GK1.5; BioLegend), anti-CD8a-APC-eFluor780 (clone 53–6.7; eBioscience), anti-CD11b-BB700 (clone M1/70; BD Horizon), anti-Ly6G-BV711 (clone 1A8; BioLegend), anti-CD25-BB515 (clone PC61; BD Horizon), anti-FoxP3-PE-Cy7 (clone FJK-16 s; eBioscience), and Ghost Dye Violet 510 (Tonbo Biosciences). Flow cytometry data were acquired using an Attune NxT Flow Cytometer and analyzed using FlowJo version 10.1.

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Voeller J., Erbe A.K., Slowinski J., Rasmussen K., Carlson P.M., Hoefges A., VandenHeuvel S., Stuckwisch A., Wang X., Gillies S.D., Patel R.B., Farrel A., Rokita J.L., Maris J., Hank J.A., Morris Z.S., Rakhmilevich A.L, & Sondel P.M. (2019). Combined innate and adaptive immunotherapy overcomes resistance of immunologically cold syngeneic murine neuroblastoma to checkpoint inhibition. Journal for Immunotherapy of Cancer, 7, 344.

Publication 2019

DNase I anti-CD45-eF450 (clone 30-F11; Ghost Dye Violet 510 Attune NxT Flow Cytometer

Anti cd3 Cd11b Cd25 Cells Clone Collagenase Dnase Flow cytometry Ghost Hbss Tumors Violet

Corresponding Organization :

Other organizations : Highland Community College - Illinois, University of Wisconsin Carbone Cancer Center, University of Wisconsin–Madison, Shriners Hospitals for Children - Philadelphia, Children's Hospital of Philadelphia

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Variable analysis

independent variables

Dissociation solution containing HBSS supplemented with 5% FBS, 1 mg/mL collagenase type D, and 100 μg/mL DNase I

dependent variables

Cell surface staining of GD2, CD45, CD3, CD4, CD8a, CD11b, Ly6G, CD25, and FoxP3

control variables

Incubation time (30 min)
Incubation temperature (37 °C)

controls

Positive control: Not specified
Negative control: Not specified

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