PE Rat Anti-Mouse CD103

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For staining of mouse lymph nodes, cells were prepared by rapidly passing the whole node through a 40 um mesh, then stained using short incubation times (10 min on ice), as described (Sharma et al., 2013).

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For staining of mouse lymph nodes, cells were prepared by rapidly passing the whole node through a 40 um mesh, then stained using short incubation times (10 min on ice), as described (Sharma et al., 2013).

Publication protocol

"The conjugated antibodies used for flow cytometry are listed in the Key Resources Table. For staining of mouse lymph nodes, cells were prepared by rapidly passing the whole node through a 40 um mesh, then stained using short incubation times (10 min on ice), as described (Sharma et al., 2013). Tumors were disaggregated by treating for 1 hr with collagenase, DNAse and hyaluronidase in RPMI 1640 medium, as described (Sharma et al., 2015).
Mouse CD11c was detected with clone HL3, to minimize cross-reactivity with macrophages. Since the available antibodies for human XCR1 have not been validated for in vitro diagnostic use, two different clones were used for confirmation (clone RTK2758 from BioLegend, and clone 1097A from R&D systems); both gave identical staining results, so the marker appeared informative.

For intracellular IL-12 detection we used antibody against the p40 subunit (clone C17.8) (Ruffell et al., 2014). There was no evidence of a role for IL-23 in our system, and results were confirmed using exogenous recombinant IL-12.

All intracellular antigens except for phospho-p53 were detected using fixation-permeabilization reagent and matching perm-wash buffer from eBioscience (Cat. #00-5523), with blocking using 5% normal donkey serum, then acquired immediately after staining. IL-12 and IFNγ were measured after 4 hr activation with PMA+ionomycin in the presence of brefeldin A as previously described (Sharma et al., 2013).

Reactive oxygen species (ROS) were measured using the redox-responsive dye 2′,7′-dichlorofluorescein diacetate (DCFDA) as described (Thevenot et al., 2014).

Antibody against the N-terminus of mouse p53 (clone 1C12) was from Cell Signaling Technology. Antibody against the C-terminus of p53 (clone PAb122) was from Novus Biologicals. For both antibodies, staining was performed following permeabilization with eBioscience fix-and-perm. Anti-phospho-p53(Ser15) (clone D4S1H) was from Cell Signaling Technology. For phospho-specific staining, cells were washed in PBS, fixed with 2% paraformaldehyde for 10 min at 37°C, pre-chilled for 1 min, then permeabilized by slow addition of ice-cold methanol to a final concentration of 90%. Cells were then incubated on ice for 30 min, washed with 1% FCS in PBS, blocked with the same solution for 10 min at room temperature, then for 1 h at room temperature and washed. Cells were acquired immediately after staining."

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