Flow cytometry analysis software

For the in vitro uptake experiments bone marrow-derived DCs (BMDCs) were generated based on a modified protocol described previously [21 (link)]. BMDCs were pulsed with OVA-FITC (Invitrogen) for different time points and uptake was measured by FACS (FACS CANTO Becton Dickinson) and analyzed with flow cytometry Analysis software (Tree Star).

As described before [22 (link)] ears were split into dorsal and ventral halves and floated split side down in 1 ​ml medium containing RPMI, 10% FCS, 50 ​μM β-mercaptoethanol and with or without CCL21 for 48 ​h. Emigrated non-adherent cells were stained with anti-CD11c (Pe-Cy7 eBioscience), anti-MHCII (APC-Cy7, Biolegend), anti-CD11b (Alexa700, eBioscience) and anti-CD103 (Percp-Cy5.5, Biolegend) and were quantified by FACS to determine the number and different subsets of emigrated DC from the skin to the culture medium. Acquisition was done using a FACS CANTO (Becton Dickinson) and data was analyzed with flow cytometry Analysis software (Tree Star).

For the in vivo experiments 10 ​μg or 100 ​μg OVA-FITC labeled (Invitrogen) was injected in the base of the tail on the right and left sides of WT and Flt3L^−/- mice and LNs were collected after 36 ​h. FITC⁺ cells were quantified using the following markers: anti-CD11c (Pe-Cy7 eBioscience), anti-MHCII (APC-Cy7, Biolegend), anti-CD11b (Alexa700, eBioscience) and anti-CD103 (Percp-Cy5.5, Biolegend). Flow cytometry was performed using a FACS CANTO (Becton Dickinson) and analyzed with flow cytometry Analysis software (Tree Star).