Utricles from Lgr5-EGFP-CreERT2+ and Plp1-tdTomato+ mice were cultured with 1 mM neomycin for 24 h to damage the HCs and to re-induce the expression of Lgr5. To obtain a single-cell suspension, all tissues were gently removed from the Petri dishes (Greiner Bio-one) using fine forceps and subjected to 0.125% trypsin at 37°C for 10 min. Soybean trypsin inhibitor was added to stop the enzymatic reaction. The cell suspension was carefully triturated with plastic pipette tips (epTIPS Filter 20– 300 μl; Eppendorf), and the cells were passed through a 40 μm cell filter (Greiner Bio-one) and collected in an Eppendorf tube. Dissociated cells were sorted on a MoFlo® SX fluorescence activated cell sorting (FACS) cytometer (Beckman Coulter, CA, USA) using the channels for GFP and tdTomato, and the positive fractions were collected for further experiments.
Eptips filter 20 300 μl
Manufactured by Eppendorf
The EpTIPS Filter 20–300 μl is a disposable pipette tip designed for laboratory use. It is suitable for volume ranges of 20 to 300 microliters. The tip features a built-in aerosol filter to help prevent contamination during pipetting.
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2 protocols using eptips filter 20 300 μl
1
Isolation of Lgr5+ and Plp1+ Utricle Cells
2
Sphere Dissociation and Propagation
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The spheres were mechanically dissociated after a 5–10 min treatment with 0.125% trypsin in PBS at 37°C in a total volume of 100 μL for each organ. The enzymatic reaction was blocked by adding 100 μL of 0.5% soybean trypsin inhibitor (Worthington) and 2 mg/mL DNAse I in DMEM/high‐glucose (4,500 mg/L dextrose) and F12 media (mixed 1:1, Invitrogen). The cells were mechanically dissected using tungsten microneedles and carefully triturated 20–50× with plastic pipette tips (epTIPS Filter 20–300 μL, Eppendorf). The cell suspensions were replated into DMEM/high‐glucose (4,500 mg/L dextrose) and F12 media (mixed 1:1) supplemented with N2 and B27 (Invitrogen), EGF (20 ng/mL), bFGF (10 ng/mL), IGF‐1 (50 ng/mL), and heparan sulfate (50 ng/mL) in Petri dishes and the propagation procedure was repeated in 7‐day‐intervals. For long‐term propagation, we counted the number of spheres for each generation, related the number to the original sphere number and replated half of the spheres for producing the next generation.
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