In cell analyzer 2000 high

Example 6

Fluorescence Imaging

Live cell wide field epifluorescent imaging was performed on a Zeiss Axio Ovserver.Z1 inverted microscope equipped with an X-Cite 120Q excitation light source using an AxioCam MRm camera and Zen Pro 2011 software (Carl Zeiss). Immuno-fluorescent (IF) binding assay of individual phage clones was performed on an IN Cell Analyzer 2000 high-content analysis system (GE Healthcare). Briefly, 10,000 antigen expressing cells were seeded into a clear 96w flat-bottom tissue culture plate one day before imaging. On the day of imaging, cells were washed once with PBS and 100 μL of purified individual phage clones in binding buffer were incubated with cells for 1 h at 20° C. Phage solutions were decanted, wells washed three times with PT buffer (PBS, 0.05% Tween 20) and fixed with 1% formaldehyde in PBS for 15 min at 20° C. After cell fixation, mouse anti-M13 phage primary antibody (1:500 in PBT, GE Healthcare) was added for 30 min at 37° C., followed by three washes with PT buffer before adding Alexa Fluor 546 Goat anti-Mouse IgG secondary antibody (1:500 in PBT, Invitrogen) for 30 min at 37° C. Nuclei were stained with Hoechst 33342 (2 μg/mL in PBT), cells were washed three times with PT buffer and placed in PBS for imaging.