mCherry signal preserved in multipotent otic progenitor cells labeled with EdU

Genetically modified induced multipotent otic progenitor cells expressing mCherry were initially cultured in DMEM/F12, B27 containing 20 ng/ml of bFGF. To promote neuronal differentiation, cells were plated onto 1.5 glass coverslips coated with 10 µg/mL poly-D-lysine and 10 µg/mL laminin and cultured in the same media for 7 days without bFGF. To incorporate the nucleoside analog EdU and identify proliferating cells, cultures were incubated with 1 µM EdU for 2 hrs before fixation. Media was removed from the cultures and cells were fixed in 1X PBS containing 4% formaldehyde for 15 minutes at RT. Cells were washed in 1X PBS containing 3% BSA and the cell membrane permeabalized in 1X PBS containing 0.5% Triton X-100 for 20 minutes at RT. To label the EdU, cells were washed with 1X. The top and bottom rows represent use of the Click-iT® Plus EdU Alexa Fluor® 488 Imaging Kit and the Click-iT® EdU Alexa Fluor® 488 Imaging Kit, respectively.

Genetically modified induced multipotent otic progenitor cells expressing mCherry were initially cultured in DMEM/F12, B27 containing 20 ng/ml of bFGF. To promote neuronal differentiation, cells were plated onto 1.5 glass coverslips coated with 10 µg/mL poly-D-lysine and 10 µg/mL laminin and cultured in the same media for 7 days without bFGF. To incorporate the nucleoside analog EdU and identify proliferating cells, cultures were incubated with 1 µM EdU for 2 hrs before fixation. Media was removed from the cultures and cells were fixed in 1X PBS containing 4% formaldehyde for 15 minutes at RT. Cells were washed in 1X PBS containing 3% BSA and the cell membrane permeabalized in 1X PBS containing 0.5% Triton X-100 for 20 minutes at RT. To label the EdU, cells were washed with 1X. The top and bottom rows represent use of the Click-iT® Plus EdU Alexa Fluor® 488 Imaging Kit and the Click-iT® EdU Alexa Fluor® 488 Imaging Kit, respectively.

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