TNF-α (315-01A) or IL-1β (211-11B) recombinant mouse peptides were purchased from PeproTech (Rocky Hills, NJ) and were resuspended in PBS. For UVB experiments, we used two TL 20W/12RS lamps (Philips, Amsterdam, The Netherlands). We used UVB doses previously described as 20 mJ/cm2 for pKCs or experiments, (Mohammed et al., 2016 (link)).
DMBA (57-97-6; Sigma-Aldrich, St. Louis, MI) was applied at 0.1mM in DMSO for pKC experiments. For in vivo mouse experiments, 10 mM of DMBA was resuspended in DMSO-to-ethanol-to-glycerol solution (1:1:3) and was applied to the flank skin of shaven mice. Antibodies directly conjugated to different fluorophores were used for flow cytometry and immunofluorescence. Anti-CD11c(N418)-PerCp5.5, CD11b(M1/70)-PeCy7, I-A/I-E/MHCII (M5/114.15.2)-AF700, Langerin(4c7)-phycoerythrin, CD103(2E7)-AF647, CD45.2(104)-BV605, Sca-1(E13-161.7)-PerCp5.5, EpCAM(G8.8)-PeCy7, and CD34(HM34)-PE/Dazzle594 were purchased from BioLegend (San Diego, CA). Viability Dye eFluor 780 (eBioscience; Invitrogen, Carlsbad, CA) was used for live‒dead discrimination. Anti-ανβ6 (6.3g9) and anti-ανβ8 (C6D4)-phycoerythrin were kindly provided by Dean Sheppard and Stephen Nishimura, respectively. Anti-ανβ6 (6.3g9) was directly conjugated to Alexa Fluor 647 (A20186; Thermo Fischer Scientific, Waltham, MA).
DMBA (57-97-6; Sigma-Aldrich, St. Louis, MI) was applied at 0.1mM in DMSO for pKC experiments. For in vivo mouse experiments, 10 mM of DMBA was resuspended in DMSO-to-ethanol-to-glycerol solution (1:1:3) and was applied to the flank skin of shaven mice. Antibodies directly conjugated to different fluorophores were used for flow cytometry and immunofluorescence. Anti-CD11c(N418)-PerCp5.5, CD11b(M1/70)-PeCy7, I-A/I-E/MHCII (M5/114.15.2)-AF700, Langerin(4c7)-phycoerythrin, CD103(2E7)-AF647, CD45.2(104)-BV605, Sca-1(E13-161.7)-PerCp5.5, EpCAM(G8.8)-PeCy7, and CD34(HM34)-PE/Dazzle594 were purchased from BioLegend (San Diego, CA). Viability Dye eFluor 780 (eBioscience; Invitrogen, Carlsbad, CA) was used for live‒dead discrimination. Anti-ανβ6 (6.3g9) and anti-ανβ8 (C6D4)-phycoerythrin were kindly provided by Dean Sheppard and Stephen Nishimura, respectively. Anti-ανβ6 (6.3g9) was directly conjugated to Alexa Fluor 647 (A20186; Thermo Fischer Scientific, Waltham, MA).