Utricles harvested from P3-P5 Lgr5EGFP-CreERT2/+; Rosa26RtdTomato/+ mice were attached to 35 mm glass bottom dishes (MatTek) pre-coated with CellTaK (BD Biosciences). Whole organs were cultured overnight, then treated with neomycin (1.0 mM × 24hr, Sigma) as above. 4OH-tamoxifen (500 nM, Sigma) in growth factor-enriched, serum-free media was present for 2–4 days first. Then organs were imaged in DMEF/F12 without phenol red (1:1 Cellgro) media using a spinning disc confocal imaging system (Zeiss Axio Observer Z1 or OlympusIX-81 coupled with a Yokogawa spinning disc system CSU-X1A 5000) connected to an incubating chamber (37°C, 5% CO2). One to two EGFP+ regions with tdTomato+ cells were selected from each utricle, and z-stack images spanning the sensory epithelium were taken at 0.5–1 hr intervals. Collected videos were processed and analyzed with ImageJ64 (NIH), MetaMorph (NX 2.0; Olympus) and Volocity software (v6.1.0; Improvision).
Metamorph nx 2.0
Manufactured by Olympus
MetaMorph (NX 2.0) is a software package designed for image acquisition, processing, and analysis. It provides a comprehensive suite of tools for researchers to capture, manage, and explore digital images from a variety of microscopy and imaging systems.
Automatically generated - may contain errors
2 protocols using metamorph nx 2.0
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Imaging Hair Cell Regeneration
2
Imaging Hair Cell Regeneration
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Utricles harvested from P3-P5 Lgr5EGFP-CreERT2/+; Rosa26RtdTomato/+ mice were attached to 35 mm glass bottom dishes (MatTek) pre-coated with CellTaK (BD Biosciences). Whole organs were cultured overnight, then treated with neomycin (1.0 mM × 24hr, Sigma) as above. 4OH-tamoxifen (500 nM, Sigma) in growth factor-enriched, serum-free media was present for 2–4 days first. Then organs were imaged in DMEF/F12 without phenol red (1:1 Cellgro) media using a spinning disc confocal imaging system (Zeiss Axio Observer Z1 or OlympusIX-81 coupled with a Yokogawa spinning disc system CSU-X1A 5000) connected to an incubating chamber (37°C, 5% CO2). One to two EGFP+ regions with tdTomato+ cells were selected from each utricle, and z-stack images spanning the sensory epithelium were taken at 0.5–1 hr intervals. Collected videos were processed and analyzed with ImageJ64 (NIH), MetaMorph (NX 2.0; Olympus) and Volocity software (v6.1.0; Improvision).
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