Single cell suspensions of collagenase-digested tumors were stained with the following antibodies purchased from eBioscience: fixable viability dye efluor 450, CD69-FITC, PD-1-PE, CD4-PeCy7, CD45-Alexa Fluor 700, CD8a-PE efluor 610, CD40-FITC, CD70-PE, MHC-II IAd-APC, CD11c-PE efluor 610, CD45-APC, CD3-PerCP-Cy5.5, CD11b-PerCP-Cy5.5, EpCAM-PeCy7, PD-L1-PE and PD-L2-FITC. Phospho-Smad2/3 levels were assessed as previously described (27 (link)). Briefly, TDLN cells were stained with anti-mouse CD4-PE and anti-mouse CD8-FITC (eBioscience), fixed, permeabilized (Foxp3 Fixation/Permeabilization Concentrate and Diluent kit, eBioscience) and stained with goat anti-phospho-Smad2/3 (Ser 423/425) followed by APC-labeled donkey anti-goat IgG (Santa Cruz Biotechnology). All samples were acquired with LSRII flow cytometer and analyzed with FlowJo software (version 7.3.6).
Protocol detail
Multiparameter Tumor Immune Profiling
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Corresponding Organization : New York University
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Variable analysis
independent variables
- Collagenase digestion of tumors
- Expression levels of CD69, PD-1, CD4, CD45, CD8a, CD40, CD70, MHC-II IAd, CD11c, CD45, CD3, CD11b, EpCAM, PD-L1, PD-L2
- Phospho-Smad2/3 levels
- Fixable viability dye efluor 450
- TDLN cells stained with anti-mouse CD4-PE and anti-mouse CD8-FITC
- Not explicitly mentioned
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