Tumors were excised at days 1, 7 and 14 after IRE. Tumor tissues were snap frozen and homogenized in gentle tissue lysis buffer (Thermo Scientific), and lysates were collected in the presence of protease and phosphatase inhibitors (Pierce Protease and Phosphatase Inhibitor Mini Tablets, Thermo Scientific). Proteome Profiler Mouse Cytokine Expression Array (R&D systems) was used to capture intratumoral cytokines according to manufacturer’s instructions. An array intensity measurement layout was created using ImageJ, and the area of the positive control spots was defined and used to define the area of test spots. All signal spots falling outside the filter layout were considered as artefacts. Chemiluminescence imtensity of the duplicate spots on the membranes was calculated using ImageJ [27 (link)]. The relative fold changes in chemiluminescence against control tumors at the same timepoints were compared.
Protocol detail
Intratumoral Cytokine Profiling of IRE-Treated Tumors
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Buffer
Chemiluminescence
Cytokines
Frozen
Inhibitors
Membranes
Mouse
Phosphatase
Protease
Proteome
Spots
Tissue
Tumors
Corresponding Organization :
Other organizations : University of California, San Diego, La Jolla Institute For Allergy & Immunology
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Variable analysis
independent variables
- Days after IRE (1, 7, 14)
- Intratumoral cytokine expression
- Tumor tissues were snap frozen and homogenized in gentle tissue lysis buffer (Thermo Scientific)
- Lysates were collected in the presence of protease and phosphatase inhibitors (Pierce Protease and Phosphatase Inhibitor Mini Tablets, Thermo Scientific)
- Proteome Profiler Mouse Cytokine Expression Array (R&D systems) was used to capture intratumoral cytokines according to manufacturer's instructions
- An array intensity measurement layout was created using ImageJ, and the area of the positive control spots was defined and used to define the area of test spots
- All signal spots falling outside the filter layout were considered as artefacts
- Chemiluminescence intensity of the duplicate spots on the membranes was calculated using ImageJ
- Positive control spots
- Not explicitly mentioned
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