Transfection and validation of BK channel expressing HEK-293 cells for the study of miR-9 regulation.

Thaw cells and then plate them as follows:

Resuspend in 1mL of modified DMEM media composed of Dulbecco’s modified Eagle’s medium (D5796, Milwaukee, WI, USA) with 10% fetal bovine serum (FBS), 2.52mM HEPES; 10 units and 10µg/mL of Penicillin/Streptomycin (Pen/Strep), and 1mM Na-pyruvate.

Pipette 20µL of the resuspended solution onto 35mm

petri dishes (surface area of 8.8c²2).

Flood with DMEM up to a final volume of 2mL.

Place cells in a Sanyo CO₂ incubator (37.0^oC/5% CO₂ level) and allow them to reach full confluency replacing the media every 3-5 days.

Once the cells reach 70-90% confluency suction media from the plates and add Trypsin-EDTA 1:10 diluted in 1X PBS to solubilize the cells. Apply gentle shaking to the plates to allow for the separation of the cells from the plate. Add a volume of DMEM to the plate equal to the added volume of trypsin to prevent further cell lysis.

Pipette suspended cells onto 10mL tubes.

Remove 10mL tubes from the chemical hood and centrifuge at 1,380g for 60 seconds.

Place the tubes inside the chemical hood and aspirate the supernatant.

Resuspend in 1mL of DMEM.

Extract 10µL of each tube and mix with equal volume of Trypan Blue Dye.

Add a single droplet of the Trypan Blue mixture to a counting slide and place in an automated cell counter.

While the cells are being counted, add an additional 9mL of modified DMEM to the tube and gently resuspend, for a total volume of 10mL.

Once counted add the appropriate seeding density (0.3x10⁶ cells) to the 35mm petri dishes and place them in the incubator and allow them to reach 70-90% confluence.

Reseed cells onto a 24-well plate and incubate until they reach 70-90% confluence.

Once cells reach 70-90% confluence remove the plates from the incubator and place them in the cell culture hood.

Wash the plates 3 times with 1X PBS for 30 seconds. Gently swirl between washes.

Add serum-deprived DMEM (no FBS) to the plates and place them in the incubator for 24 hours.

After incubation prepare a 100µL solution of Opti-MEM reduced serum media containing 0.8µg of cDNA BK ZERO isoform vector and 2.0µL of Lipofectamine 2000 (11668027, Thermofisher, NY,

USA).

Incubate mixture for 5 minutes at room temperature before use.

Add the mixture to each well in the 24-well plate and place them in the incubator for 16 hours to allow for transfection to occur.

After the incubation period, completely exchange transfection media with DMEM.

Wash 3 additional times with 1X PBS and place in modified DMEM for 24 hours in the incubator.

Reseed cells onto a 24-well plate and incubate until they reach 70-90% confluence.

Once cells reach 70-90% confluence remove the dishes from the incubator and place them in the cell culture hood.

Wash the plates 3 times with 1X PBS for 30 seconds. Gently swirl between washes.

Add serum-deprived DMEM (no FBS) to the plates and place them in the incubator for 24 hours.

After incubation prepare a 100µL solution of Opti-MEM reduced serum media containing 0.8µg of cDNA BK ZERO isoform vector and 2.0µL of Lipofectamine 2000 (11668027, Thermofisher, NY,

USA).

Incubate mixture for 5 minutes at room temperature before use.

Add the mixture to each well of the 24-well plate and place them in the incubator for 24 hours to allow for transfection to occur.

After the incubation period, completely replace the transfection media with DMEM the reduced.

Wash 3 additional times with 1X PBS and place in modified DMEM for 24 hours in the incubator.

After transfection, of BK ZERO constructs, remove cells from the incubator and wash 3 times (30 secs each) with 1X PBS. Add DMEM supplemented with 400µg/mL of G418 (Gentamicin)(Sigma Aldrich, A1720 Milwaukee, WI, USA) antibiotic.

Replace half of the media volume with modified DMEM every 2-3 days for 10-14 days or until visually resistant colonies are visible.

Transfer cells from the 24-well plate (as described in the cell culture section) and plate onto 96-well plate by limited dilution (to create monoclonal cell lines) as follows:

In a 96-well plate pipette 12μL of cell culture media into all except A1.

Pipette 200μL of the cell suspension into well A1.

Pipette 100μL from well A1 to A2 and gently resuspend gently.

Repeat dilutions from A2 to A3 and subsequently throughout the entire row.

Then Pipette 100μL from all wells on Row A too wells on row B and resuspend gently.

Repeat this process for pair of rows (B-C, C-D…).

Add 100μL of media to each well.

Incubate for 4 to 5 days.

Observe daily to ensure colonies were formed by a single cell.

Identify wells containing colonies from single-cell origin. Allow these colonies to reach 40% confluence. Subculture these colonies individually for validation of construct genomic insertion.

Stably transfected cells are plated in 35mm dishes and allowed to reach 30-50% confluence. Prior to experiments, dishes are gently washed 3 times with 1X PBS to remove any remaining fetal bovine serum from media; and then placed in Normal Locke’s solution (mM):145 NaCl, 2.5 KCl, 10 HEPES, 2 CaCl₂, 1 MgCl₂, pH 7.5.

Allow plates to stand 30 minutes before use.

Single-channel recordings are obtained in the cell-attached patch clamp mode under the following conditions:

Internal (pipette)solution (mM): 150 k-gluconate, 1 MgCl₂, 10 HEPES, PH 7.4.

External bath solution (mM): 145 NaCl, 2.5 KCl, 10 HEPES, 2 CaCl₂, 1 MgCl₂, pH 7.5.

orISO K⁺: 147 KCL, 10 HEPES, 2 CaCl₂, pH 7.5.

Transfer plates to the recording chamber and wash dishes with ISO K⁺ solution 3 to 5 times (30 secs each).

Carefully create a patch seal of 1-3GΩ and begin recording.

When using the standard bath, correct for the effective applied voltage by estimating correction deviations using the leak currents during cell-attached recordings.

Aquire Data using a HEKA EPC10 patch-clamp amplifier controlled by a Macintosh-based computer system equipped with Patch Master acquisition software.

Sample Data at 25μs per sample and filtered at 10kHz low bandpass.

Perform data analysis with IgorPro graphing and curve fitting software as follows:

Determine NP_o for each voltage using the following:

where N is the number of observed channels (levels), i the ith level number, and A_i the area of a Gaussian fit to the ith level’s in all-points-histogram from each voltage.

When seeking the half-action voltage (V_1/2) from NP_o versus voltage curves, determine the values from sigmoidal fits assuming the following:

where V_1/2 is the half-action voltage and V the applied voltage. The V_1/2's were thus, obtained from the half height of the sigmoidal fits.

For ICC cells were plated in 35mm glass bottom petri dishes (MATTEK Life Sciences,

P35G-1.5-20-C MA, USA).

Cells were allowed to reach 50-60% confluence and then removed from the incubator and then wash 3 times with 1X PBS (30 secs each).

Cell were fixed by placing plates in a 4% paraformaldehyde (PFA) solution (Santa

Cruz Biotechnology, CAS 30525-89-4 TX, USA) for 1 hour while gently rocking.

Excess PFA was removed with 3 washes using ice-cold 1X PBS.

Cells were first incubated for 1 hour with Cholera Toxin B Alexa Fluor 594 conjugate (CtB) antibody(C22842,Thermofisher, New York, NY, USA) 864ng/mL to label GM1.

Wash 3 additional times with 1X PBS.

Following the final wash, remove all 1X PBS.

Add Fluoromount-G™ Mounting Medium, with DAPI 00-4959-52 (Thermo Fisher) to the dishes prior to placing a cover slip, Premium Cover Glass 12-548-B (Fisher Scientific USA).

Image using Nikon Instruments A1 Confocal Laser Microscope with 60X objective (Refractive Index=1.51 and Numerical Aperture=1.40) (Maintain imaging parameters between treatments).

Analyze Mean intensity fluorescence and colocalization by Nikon Software NIS Elements v5.30 – Software, Automated Measurements – Module.