Fluorescent Peptide Localization in Cells

The NAF-1^44–67 peptide was labeled with 5(6)-carboxyfluorescein at its N-termini as described in [15 (link)]. In a 96 well plate, 15,000 cells per well were allowed to grow for three days prior to experiments. Vesicles and PM were labeled with 5 µM of SynaptoRed^TM C2 in DMEM cell culture medium for 1 h and washed with DMEM cell culture media. The cells were then incubated in phenol red free DMEM with 100,000 U/ml Penicillin/Streptomycin with 15 µM of the peptide (Fl-NAF-1^44–67). The cells were imaged using a BioTek Lionheart FX (https://www.biotek.com) apparatus at 37 °C, 5% CO₂ under humidified condition using the following wavelengths 586/647 nm for the SynaptoRed^TM C2 and 469/525 nm for Fl-NAF-1^44–67. Image analysis and quantification were conducted using ImageJ (https://www.nih.gov). Briefly, the fluorescence signal of Fl- NAF-1^44–67 peptide was overlapped with phase contrast images and the intensity of signal was then discriminated between PM and intracellular localization.