Fm1 43

FM1-43 was performed as described previously (60 ). Melanoma cells were left unpulsed or pulsed with 10 μM antigenic peptide (or lower concentrations when indicated) for 2 hours at 37°C/5%CO₂, washed three times, and subsequently transferred to a 96-well U-bottom plate at 20 × 10³ cells per 25 μl of RPMI 1640 5% FCS/Hepes. CTLs were previously stained with CTV (Invitrogen) for 20 min at 37°C/5% CO₂, washed, and added to the target cells at a two CTL/one target cell ratio in 25 μl of RPMI 1640 5% FCS/Hepes. Fifty microliters of FM1-43 (20 μg/ml) (Invitrogen) in RPMI 1640 5% FCS/Hepes was added in 50 μl of cells already contained in the well (FM1-43 final concentration was 10 μg/ml). In some experiments, D10 cells were pretreated with 50 μM BAPTA-AM for 2 hours at 37°C (Invitrogen) and thoroughly washed before conjugation with CTLs. Cells were pelleted for 1 min, 455g and incubated at 37°C/5% CO₂ for 2, 5, or 15 min. At the end of each incubation time, CTL/melanoma cell cocultures were resuspended and washed in ice-cold phosphate-buffered saline (PBS) containing 2 mM EDTA to dissociate CTL/melanoma cell conjugates. Cells were stained with fixable viability dye eFluor 780 (eBioscience) on ice at 4°C for 30 min in FACS buffer (PBS 1% HS, 1% FCS, and 0.01% azide). Samples were acquired using MACS Quant Analyzer 10 (Miltenyi Biotec). Results were analyzed using the FlowJo 10 software.

Exocytosis was assessed by real‐time imaging using the fluorescent dye, FM1‐43 (Molecular Probes, Inc., Eugene, OR), as described.19 FM1‐43 is weakly fluorescent in aqueous solution, but its fluorescence increases >300‐fold when it binds plasma membrane; thus, it is a useful dye for the measurement of increases in plasma membrane attributed to fusion of vesicle membrane with the plasma membrane during exocytosis. Changes of FM1‐43 fluorescence were measured at excitation wavelength 480 nm and emission wavelength at 535 nm and reported as % change in fluorescence.

Cochleae were isolated from Atoh1^Cre/+;Insm1^F/F at P5 and explants set up as described above. After 6 days in vitro, explants were washed in HBSS (14025076, Thermo Fisher Scientific). Once in wash, a 10 μM FM1-43 (T3163, Thermo Fisher Scientific) working solution was prepared in HBSS. The HBSS wash was removed, and the FM1-43/HBSS solution was added and allowed to sit for 20 s. Cultures were then washed three times in HBSS and immediately examined for fluorescence with a Nikon E600 upright fluorescence microscope [first with a Plan Apo 10× lens (with a numerical aperture of 0.45) to locate the tissue and then with a Fluor 40× immersion lens (with a numerical aperture of 0.8 W)]. Images were captured using a Nikon DS-Ri2 Color complementary metal-oxide semiconductor camera. Cultures were then fixed in 4% PFA in PBS for 30 min at room temperature and processed for immunostaining and confocal microscopy, as described above, to label the oc-IHCs.