N formyl nle or leu phe nle tyr lys fluorescein

Cells (~2×10⁴/sample) were seeded on glass coverslips and cultured for 24 hrs in growth medium. Then, slides were washed with PBS, fixed with 2.5% formaldehyde in PBS for 10 min at 4°C and incubated overnight at 4°C with 2 μg/mL R4 anti-uPAR monoclonal antibody or rabbit anti-uPAR_84-95 polyclonal antibody [30 (link)]. HPMCs plated on glass slides (30%-40% confluence), were fixed and permeabilized with 2.5% formaldeyde-0.2% Triton X-100 in PBS for 10 min at 4°C, then incubated with 2 μg/mL anti-vimentin monoclonal antibody (Dako), anti-cytokeratin 8/18 polyclonal antibody (MyBiosurce), or anti-von Willebrand factor VIII monoclonal antibody (Dako) for 1 hr at 4°C. Then, 1:800 goat Alexa Fluor 488 anti-rabbit IgG, Alexa Fluor 488-conjugated F(ab')2 fragment of rabbit anti-mouse IgG, or Alexa Fluor 594 goat anti-mouse IgG (Molecular Probes) were applied to slides at 23°C for 40 minutes. After nuclear staining with 4-6-diamidino-2-phenylindole dye (DAPI), coverslips were mounted using 20% (w/v) mowiol and analyzed by a fluorescence inverted microscope connected to a video-camera (Carl Zeiss). To analyze FPR internalization, cells grown on glass slides were exposed to 10 nM N-formyl-Nle-or Leu-Phe-Nle-Tyr-Lys-fluorescein (Molecular Probes), diluted in serum-free DMEM for 30 minutes at 37°C as described [32 (link)].

Cells (~2 × 10⁴/sample) were seeded on glass coverslips and cultured for 24 h in growth medium. Then, slides were washed with PBS, fixed with 2.5% formaldehyde in PBS for 10 min at 4 °C and incubated for 1 h at 4 °C with 2 μg/mL R4 anti-uPAR monoclonal antibody or rabbit anti-1:100 anti-FPR1 antibody (#113531Ab, Abcam). Then, 1:700 goat Alexa Fluor 488 anti-rabbit IgG or rabbit Alexa Fluor 488-conjugated F(ab’)2 fragment of anti-mouse IgG (Molecular Probes) were applied to slides at 23 °C for 40 min. Nuclear staining was performed with 4–6-diamidino-2-phenylindole dye (DAPI). To visualize the cytoskeleton, cells were fixed with 2.5% formaldehyde, permeabilized with 0.1% Triton X-100 for 10 min at 4 °C, and incubated with 0.1 μg/mL rhodamine-conjugated phalloidin (Sigma-Aldrich) for 40 min. To analyze agonist-dependent FPR1 internalization, cells grown on glass slides were exposed to 10 nM N-formyl-Nle-or Leu-Phe-Nle-Tyr-Lys-fluorescein (Molecular Probes), diluted in serum-free DMEM for 30 min at 37 °C as described [39 (link), 40 (link)]. In all cases, coverslips were mounted using 20% (w/v) Mowiol, visualized with the the Axiovert 200 M fluorescence inverted microscope connected to a video-camera or with the 510 META-LSM confocal microscope (Carl Zeiss).