Moflo sx facs cytometer

The isolation of HC progenitors was performed as in previous studies (Zhang et al., 2015 (link); Waqas et al., 2016 (link); Cheng et al., 2017 (link)). Briefly, postnatal day (P)0–P3 mouse sensory epithelium samples were digested by 0.125% trypsin/EDTA (Invitrogen) in 0.01M PBS for 9 min at 37°C. The reaction was terminated by soybean trypsin inhibitor (10 mg/ml; Worthington Biochem). Following mechanical trituration with about 80–100 strokes with blunt tips (Eppendorf, #22491245), isolated cells were percolated through a 40 μm cell strainer (BD Biosciences). Non-viable cells were labeled by propidium iodide (1 μg/ml, Sigma). Dissociated cells were then sorted using the GFP channel on a MoFlo^® SX FACS cytometer (Beckman Coulter), and the EGFP+ cells were collected. Isolated cells were lysed in RLT buffer (Qiagen) for quantitative real-time PCR (q-PCR) assay or stored at -80°C until RNA-Seq analysis. For each RNA-Seq library, nearly 10,000 sorted cells were used. Re-sort analysis, immunofluorescence, and q-PCR were used to confirm the identity of the sorted cells.

Cochleae from P3 Lgr6-EGFP-Ires-CreERT2 mice were dissected out and the stria vascularis and spiral ganglia were removed. The cochleae were incubated in 0.125% trypsin (Invitrogen) at 37°C for 8 min, and the same volume of 10 mg/mL trypsin inhibitor (Worthington Biochem) was added. Following trituration, cells were passed through a 40 μm filter (BD Biosciences) and labeled with 1 μg/mL propidium iodide (Sigma) to remove the dead cells. Wild-type cochleae were used to determine the background labeling levels in each sorting. The dissociated cells were sorted on a MoFlo® SX FACS cytometer (Beckman Coulter) using the channels for GFP, and the positive fractions were collected. We consistently achieved over 90% cell viability and over 95% purity for sorted cells. The purity of the sorted cells was assessed by re-sort analysis, immunohistochemistry, and quantitative RT-PCR. For staining, sorted cells were plated on fibronectin-coated slides (Sigma) and incubated for 1 h at 37°C before fixation and immunohistochemistry.