dos.5. Ca dos+ imaging when you look at the neonatal OHCs

The fresh rat Ocm gene (series for the Profile S01) was cloned towards pcDNA3.1CFP and you may pEGFP-N1 Banner (Addgene #60360). Following, EGFP-Flag try eliminated using BamHI (NEB) and NotI (NEB) and you can substituted for the mCherry series (gene cut-off off mCherry regarding Qiagen) to help make pOcm-mCh. Rodent Apv and you can Sri (custom ordered gene stop from IDT, Coralville, IA) (sequences from inside the Profile S01) have been switched into Ocm webpages having fun with HindIII (NEB)/BamHI or HindIII/SalI to make pApv-mCh and you may pSri-mCh, correspondingly. The mCherry plasmid along with changes the fresh new Banner linker that have a flexible GS linker (SGGGGSGGGGSGGGGS) between Ocm/Apv/Sri and mCherry. Brand new mCh control plasmid is actually generated by cloning out of the Ocm gene away from Ocm-mCherry. Nucleotide sequences to own rodent Ocm/Apv/Sri and plasmid sequences to possess pOcm-CFP, pmCh, pOcm-mCh, pApv-mCh and you may pSri-mCh can be found in Shape S01.

dos.3. Cell People and Reagents

Human Embryonic Kidney (HEK293T) cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM, Thermo Fisher) supplemented with 10% fetal bovine serum (FBS, Bio-techne) at 37 ? C in a 5% CO₂ incubator. Transfections were performed using Lipofectamine 3000 (Thermo Fisher Scientific) in Opti-Mem reduced serum media (Thermo Fisher) according to manufacturer’s protocol. Mammalian cells were transfected with either mCherry (mCh), Apv-mCh, Ocm-mCh, Sri-mCh, or Ocm-CFP plasmids. 24 hours post transfection, growth media was replaced with HBSS (Gibco) before Ca 2+ transient experiments.

dos.4. Single-cell electrophysiology

OHCs from CBA/CaH Ocm +/ – and Ocm – /- mice of both sexes were acutely dissected at P2 and P3. Cochleae were isolated female escort Arvada CO from the inner ear as previously reported , , , using an extracellular solution composed of (in mM): 135 NaCl, 5.8 KCl, 1.3 CaCl₂, 0.9 MgCl₂, 0.7 NaH₂PO₄, 5.6 D-glucose, 10 HEPES-NaOH. Sodium pyruvate (2 mM), amino acids and vitamins were added from concentrates (Thermo Fisher Scientific, UK). The pH was adjusted to 7.5 (osmolality ?308 mmol kg – 1 ). The dissected cochleae were fixed at the bottom of the recording chamber by a nylon-meshed silver ring and perfused with the above extracellular solution. OHCs were viewed using an upright microscope (Olympus BX51) equipped with Nomarski Differential Interface Contrast (DIC) optics with a 60x water immersion objective and 15x eyepieces.

Recordings were performed at room temperature (21-24°C) using an Optopatch amplifier (Cairn Research Ltd, UK). Patch pipettes were pulled from soda glass capillaries and the shank of the electrode was coated with surf wax (2–3 M?). Current and voltage responses were measured using the following intracellular solution: 145 mM KCl, 3 mM MgCl₂, 1 mM EGTA-KOH, 5 mM Na₂ATP, 5 mM HEPES-KOH, 10 mM sodium phosphocreatine (pH adjusted to 7.28 with KOH; osmolality was 294 mmol kg – 1 ). Voltage clamp protocols are referred to a holding potential of -84 mV. Data acquisition was performed using pClamp software (Axon Instruments, Union City, CA, USA) using a Digidata. Data were filtered at 2.5 kHz (8-pole Bessel), sampled at 5 kHz, and stored on computer. Offline data analysis was performed using Origin software (OriginLab, Northampton, MA, USA). Membrane potentials reported were corrected for the uncompensated residual series resistance (R_s) and the liquid junction potential (LJP), which was -4 mV, measured between electrode and bath solutions.

Three procedures were used to measure California dos+ character inside neonatal OHCs. To have proportions using California dos+ signal dye Fluo-4, California dos+ transients were caused both from the local applying of KCl otherwise thru KCl superfusion. Getting proportions having fun with ratiometric California dos+ indicator color Fura-dos, Ca dos+ transients was basically triggered via KCl superfusion. For everybody three tips, organ regarding Corti spirals was in fact prepared because the described in earlier times [29, 31]. The latest apical 3rd of your cochlea, equal to the fresh 6-12 kHz part regarding mature , was used.