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

Thaw and plate the cells as follows:

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

Pipette 20µL of the resuspended solution onto 35 mm

petri dishes.

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

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

Once the cells are fully confluent suction media from the plates and add Trypsin-EDTA 10X diluted in PBS to solubilize the cells.

Add an equal volume of modified DMEM to prevent cell lysis.

Pipette suspended cells onto 10mL tubes.

Remove 10mL tubes from the hood and centrifuge at 3,300rpm for 60 seconds.

Place inside the hood and aspirate the supernatant.

Resuspend in modified 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.

Once counted add the appropriate seeding density ( 50- 80% confluent ) was to the 35 mm plate to being transfections.

Once cells reach 90% confluence place the plates in the hood.

Wash the plates 3 times with PBS for 30 seconds with gentle swirling.

Place the cells in serum deprived DMEM (no FBS) for 24 hours in the incubator.

The next day prepare solution containing the DNA of interest by diluting DNA in 250ml of Opti-MEM I - Reduced Serum Medium, mixing gently, adding

lipofectamine 2000, then diluting the appropriate amount in 250μl of Opti-MEM I Medium, and incubating for 5 minutes at room temperature. See attached Protocol.

After the incubation period syphon the serum deprived media from the plates.

Wash three time (30sec each) with PBS and gentle swirling.

After the washes Add Opti-MEM I solution for 16 hours.

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

After transfections the cells wash 3 times with PBS and place in modified DMEM supplemented with 400µg/mL of G418 antibiotic.

Replaced half of the volume with modified DMEM for 10-14 days or until visually resistant colonies where visible.

Plate onto 96-well plate by limited dilution (to create monoclonal cell lines) as follows:

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

Pipette 200 μL of the cell suspension into well A1.

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

Repeat this process for the entire row.

Then Pipette 100 μL 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 100 μL of media to each well.

Incubate for 4 to 5 days.

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

Allow cells to reach 40% confluence at which point they were expanded for storage in liquid nitrogen or for use in experiments.

Remove transfected cells from the incubator and place in Normal Locke’s media.

Then perform single channel recordings in the cell-attached mode using the following conditions:

External bath solution: 145 NaCl, 2.5 KCl, 10 HEPES, CaCl₂, 1MgCl_2.

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

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 25 μs per sample and filtered at 10kHz low bandpass

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

Determine NPo 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 NPo versus voltage curves, determine the values were 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 place plates in the hood and wash 3 times with PBS as previously described.

Fix in a 4% paraformaldehyde (PFA) solution for 1 hour with gentle shaking.

Remove excess PFA with 3 washes with ice cold PBS.

Incubate for 1 hour with Cholera Toxin B Alexa Fluor 594 conjugate (CtB) antibody 864ng/mL to label GM1.

Wash 3 additional times with PBS.

Following the final wash, remove all PBS.

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

Image using Nikon Instruments A1 Confocal Laser Microscope using a 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.