TITLE: Transfection and validation of BK channel expressing HEK-293 cells for the study of Mir-9 regulation.
AUTHORS: Katherine Cordero Padilla, Gerardo L. Alvarado Monefledt, Adriel Guevarez-Galan, Hector G Marrero Hernandez, Mario E. Lloret-Torres, Cristina Velazquez Marrero

[DESCRIPTION]
Research has identified the large conductance voltage- and calcium-activated potassium channel (BK) as a key regulator of neuronal excitability genetically associated to behavioral alcohol tolerance. Sensitivity to ethanol at the molecular level is characterized by acute potentiation of channel activity. BK isoforms show variations in alcohol sensitivity and are differentially distributed on the plasma membrane surface in response to prolonged exposure. MiRNA targeting of alcohol sensitive isoforms coupled with active internalization of BK channels in response to ethanol are believed to be key in establishing homeostatic adaptations that produce persistent changes within the plasma membrane of neurons. In fact, MiR-9 upregulated expression is a key event in persistent alcohol tolerance mediating acute EtOH desensitization of BK channels. The exact nature of these interactions remains a current topic of discussion. To further study the effects of Mir-9 on the expression and distribution of BK channel isoforms we designed an experimental model by transfecting human BK channel isoforms ZERO and STREX heterologous constructs in HEK293 cells respectively expressing 2.1 (Mir-9 responsive), 2.2 (unresponsive) and control (no sequence) 3’UTR miRNA recognition sites. We used imaging techniques to characterize the stably transfected monoclonal cell lines, and electrophysiology to validate channel activity. Finally, we used immunocytochemistry to validate isoform responsiveness to MiR-9. Our findings suggest the cell lines were successfully transfected and have differential responses to miR-9 making them viable for use in future alcohol dependence studies.


[GUIDELINES]
All cell culture procedures were performed inside a biological hood using a sterile cell culture technique. HEK-293 is a cell line exhibiting epithelial morphology that was isolated from the kidney of a human embryo commercially available through ATCC (American Type Culture Collection) a nonprofit, global biological resource center.

[STEPS]
SECTION: Cell Culture
1. Thaw and plate the cells as follows:
SECTION: Cell Culture
1.1. 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.
SECTION: Cell Culture
1.2. Pipette 20µL of the resuspended solution onto 35 mm
petri dishes.
SECTION: Cell Culture
1.3. Flood with modified DMEM up to a final volume of 2mL.
SECTION: Cell Culture
2. Place cells in a Sanyo CO2 incubator (37.0oC/5% CO2 level) and allowed to reach full confluency changing the media every 3-5 days.
SECTION: Cell Culture
3. Once the cells are fully confluent suction media from the plates and add Trypsin-EDTA 10X diluted in PBS to solubilize the cells.
SECTION: Cell Culture
4. Add an equal volume of modified DMEM to prevent cell lysis.
SECTION: Cell Culture
5. Pipette suspended cells onto 10mL tubes.
SECTION: Cell Culture
6. Remove 10mL tubes from the hood and centrifuge at 3,300rpm for 60 seconds.
SECTION: Cell Culture
7. Place inside the hood and aspirate the supernatant.
SECTION: Cell Culture
8. Resuspend in modified DMEM.
SECTION: Cell Culture
9. Extract 10µL of each tube and mix with equal volume of Trypan Blue Dye.
SECTION: Cell Culture
10. Add a single droplet of the Trypan Blue mixture to a counting slide and place in an automated cell counter. 
SECTION: Cell Culture
11. Once counted add the appropriate seeding density (50- 80% confluent) was to the 35 mm plate to being transfections. 
SECTION: Transfection
12. Once cells reach 90% confluence place the plates in the hood.
SECTION: Transfection
13. Wash the plates 3 times with PBS for 30 seconds with gentle swirling.
SECTION: Transfection
14. Place the cells in serum deprived DMEM (no FBS) for 24 hours in the incubator.
SECTION: Transfection
15. 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.
SECTION: Transfection
16. After the incubation period syphon the serum deprived media from the plates.
SECTION: Transfection
17. Wash three time (30sec each) with PBS and gentle swirling.
SECTION: Transfection
18. After the washes Add Opti-MEM I solution for 16 hours.
SECTION: Transfection
19. Wash 3 additional times with PBS and place in modified DMEM for 24 hours in the incubator.
SECTION: Monoclonal Selection
20. After transfections the cells wash 3 times with PBS and place in modified DMEM supplemented with 400µg/mL of G418 antibiotic.
SECTION: Monoclonal Selection
21. Replaced half of the volume with modified DMEM for 10-14 days or until visually resistant colonies where visible.
SECTION: Monoclonal Selection
22. Plate onto 96-well plate by limited dilution (to create monoclonal cell lines) as follows:
SECTION: Monoclonal Selection
22.1. In a 96 well plate pipe 12 μL of cell culture media into all except A1.
SECTION: Monoclonal Selection
22.2. Pipette 200 μL of the cell suspension into well A1.
SECTION: Monoclonal Selection
22.3. Pipette 100 μL from well A1 to A2 and gently resuspend gently.
SECTION: Monoclonal Selection
22.4. Repeat this process for the entire row.
SECTION: Monoclonal Selection
22.5. Then Pipette 100 μL from all wells on Row A too wells on row B and resuspend gently.
SECTION: Monoclonal Selection
22.6. Repeat this process for pair of rows (B-C, C-D…).
SECTION: Monoclonal Selection
22.7. Add 100 μL of media to each well.
SECTION: Monoclonal Selection
22.8. Incubate for 4 to 5 days. 
SECTION: Monoclonal Selection
23. Observe daily to ensure colonies were formed by a single cell.
SECTION: Monoclonal Selection
24. Allow cells to reach 40% confluence at which point they were expanded for storage in liquid nitrogen or for use in experiments.




 
SECTION: Electrophysiology
25. Remove transfected cells from the incubator and place in Normal Locke’s media.
SECTION: Electrophysiology
26.     Then perform single channel recordings in the cell-attached mode using the following conditions:
SECTION: Immunocytohemistry (ICC)
32. For ICC place plates in the hood and wash 3 times with PBS as previously described. 
SECTION: Immunocytohemistry (ICC)
33. Fix in a 4% paraformaldehyde (PFA) solution for 1 hour with gentle shaking.
SECTION: Immunocytohemistry (ICC)
34. Remove excess PFA with 3 washes with ice cold PBS.
SECTION: Immunocytohemistry (ICC)
35. Incubate for 1 hour with Cholera Toxin B Alexa Fluor 594 conjugate (CtB) antibody 864ng/mL to label GM1.
SECTION: Immunocytohemistry (ICC)
36. Wash 3 additional times with PBS.
SECTION: Immunocytohemistry (ICC)
37. Following the final wash, remove all PBS.
SECTION: Immunocytohemistry (ICC)
38. 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).
SECTION: Immunocytohemistry (ICC)
39. 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). 
SECTION: Immunocytohemistry (ICC)
40. Analyze Mean intensity fluorescence and colocalization by Nikon Software NIS Elements v5.30 – Software, Automated Measurements – Module. 
SECTION: Electrophysiology Data Analysis
31. When seeking the half-action voltage (V1/2) from NPo versus voltage curves, determine the values     were from sigmoidal fits assuming the following: 
 
where V1/2 is the half-action voltage and V the applied voltage. The V1/2's were thus obtained from the half height of the sigmoidal fits.

SECTION: Electrophysiology Data Analysis
30.1. Determine  NPo for each voltage using the following:
   
where N is the number of observed channels (levels), i the ith level number, and Ai the area of a Gaussian fit to the ith level’s in all-points-histogram from each voltage.
SECTION: Electrophysiology Data Analysis
30. Perform data analysis with IgorPro graphing and curve fitting software as follows:
SECTION: Electrophysiology
26.2. Internal (pipette) conditions: 150 k-gluconate, 1MgCl2, 10 HEPES, PH 7.4.
SECTION: Electrophysiology
27. Correct for the effective applied voltage by estimating correction deviations using the leak currents during cell-attached recordings
SECTION: Electrophysiology
26.1. External bath solution: 145 NaCl, 2.5 KCl, 10 HEPES, CaCl2, 1MgCl2.
SECTION: Electrophysiology
28. Aquire Data using a HEKA EPC10 patch-clamp amplifier controlled by a Macintosh-based computer system equipped with Patch Master acquisition software.
SECTION: Electrophysiology
29. Sample Data at 25 μs per sample and filtered at 10kHz low bandpass