Anti yfp1 e385

Recombinant human CCL19 and CCL21 were purchased from PeproTech (Rocky Hill, CT, USA), ionomycin and the chemical isoproterenol hydrochloride were obtained from Sigma-Aldrich (St. Louis, MO, USA), fluo-3-AM was from Molecular Probes (Eugene, OR, USA), whereas the luminescence substrate coelenterazine H (2-(4-Dehydroxy) coelenterazine) was purchased from Biosynth (Staad, Switzerland). All restriction enzymes were purchased from Thermo Fischer Scientific (Waltham, MA, USA). The following antibodies were used for immune fluorescence: anti-YFP1 (E385) (abcam, Cambridge, United Kingdom), anti-YFP2 (11814460001) (Roche, Basel, Switzerland), goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody Alexa Fluor^® 568 (A11011) (Invitrogen, Carlsbad, CA, USA) and goat anti-mouse IgG (H+L) cross-adsorbed secondary antibody Alexa Fluor ^® 647 (A21235) (Life Technologies, Carlsbad, CA, USA). For flow cytometry analysis of CCR7 endocytosis anti-human CCR7 APC-conjugated antibody (FAB197A) (R&D Systems, Minneapolis, MN, USA) and IgG2A APC-conjugated antibody, as isotype-matched control, (IC003A) (R&D Systems) were used.

For immunofluorescence microscopy, HEK293 cells stably expressing CCR7-YFP2 grown on coverslips were transiently transfected with individual Nb-YFP1 clones. The subsequent day, cells were stimulated or not with 0.5 µg/mL CCL19, washed and fixed in 4% formaldehyde for 15 min at RT. Afterwards, cells were permeabilized using 0.2% Triton-X-100 and 0.125 % SDS in PBG (3% BSA and 20 mM Glycine in PBS) for 30 min at RT and incubated with anti-YFP1 (E385) (abcam, Cambridge, United Kingdom) and anti-YFP2 (11814460001) (Roche, Basel, Switzerland) specific primary antibodies in PBG for 1 h at RT. Cells were washed with PBS and incubated with Alexa Fluor-labeled secondary antibodies (Invitrogen, Life Technologies) for 30 min at RT. Coverslips were mounted using Dako Fluorescence Mounting Medium (Dako, Glostrup, Denmark). Confocal images were acquired on a Leica TCS SP5 II laser scanning microscope using a 63x/1.4 NA oil-immersion objective (Leica, Wetzlar, Germany).