TITLE: TBK1 knockdown and rescue in Hela-M cells
AUTHORS: OLIVIA  HARDING, Olivia Harding, Erika L.F: Holzbaur

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">TANK-binding kinase 1 (TBK1) is a multifunctional kinase with roles in several crucial cell processes, including innate immune response, anti-viral response, and mitochondrial clearance, the last of which is of most interest to us. In order to further understand the role of TBK1 in mitophagy, we developed a protocol to transiently deplete TBK1 from a model system, HeLa cells, and re-introduce a tagged TBK1 along with other relevant components of mitophagy. Because of the many processes that rely on TBK1, cells depleted of the kinase exhibit poor health. Our protocol accomplishes ~70% depletion of endogenous TBK1 within 24 hours without causing excessive cell death. We employ this protocol to carry out biochemistry experiments such as Western blotting and organelle fractionation, and imaging experiments such as immunofluorescence, and live cell imaging.</div></div>

[STEPS]
?. [Day 1: Plating]
Trypsinize Hela-M cells by aspirating all media from a 10 cm dish of confluent cells, then dropping  onto cells.
[Trypsin]
?. [Day 1: Plating]
Incubate cells at ,  for.
37 °C
[CO2]
?. [Day 1: Plating]
Resuspend detached cells and neutralize Trypsin with  with  and  for a final volume of .
[DMEM]
[FBS]
[GlutaMAX]
1.75 mL
?. [Day 1: Plating]
Transfer this volume to 10 mL conical tube.
?. [Day 1: Plating]
Combine  with  in a 1.5 mL tube.
[of suspension]
[Trypan blue]
?. [Day 1: Plating]
Drop  onto a Countess slide and insert into the cell counter to calculate the concentration of cells in the resuspended solution.
[of this mixture]
?. [Day 1: Plating]
Plate ~0.25 million HeLaM cells on 35mm imaging dish in .
[DMEM/ FBS/ GlutaMAX]
The number of cells to plate should be calculated by a standard guide for confluence, in order to achieve ~80-90% confluence on Day 2.
I prefer to pipet the volume of suspension needed (usually 20-50 µL) for each dish directly from that 10 mL tube and add it dropwise to 2 mL in the dish. I found that the cells uptake the exogenous nucleic acids best when they are in confluent patches instead of evenly distributed on the surface.
?. [Day 2: Transfection]
Examine cells by compound microscope  -  after plating to confirm 80-90% confluence.
If cells are not at 80-90% confluence, do not transfect. Wait until they reach 80-90%.
?. [Day 2: Transfection]
For each dish, prepare the following two solutions in 1.5 mL tubes.Tube 1 (nucleic acids): +  (stock at 1µg/µL)+ (“ “)+ (“ “)+ + Tube 2 (Lipofectamine 2000): +
[Opti-mem]
[Halo-TBK1]
[mito-dsRed]
[Parkin]
[siTBK1-5’]
[siTBK1-3’]
Invert stock solutions of each plasmid several times in order to ensure even distribution of plasmid.
[Opti-mem]
[Lipofectamine 2000]
The Lipofectamine 2000 volume is calculated by this equation:
?. [Day 2: Transfection]
Invert tubes 8 times to distribute the contents, then:
?. [Day 2: Transfection]
Incubate  - at  .
0 Room temperature
?. [Day 2: Transfection]
Spin  in a minicentrifuge.
?. [Day 2: Transfection]
Add Tube 2 to Tube 1 and invert 8 times to mix.
?. [Day 2: Transfection]
Incubate  -  at .
37 Room temperature
?. [Day 2: Transfection]
Spin  in a minicentrifuge.
?. [Day 2: Transfection]
Add entire volume (~>) to the cells dropwise, distributing the drops mostly in the center of the dish (where the imaging window/coverslip is).
400 µl
?. [Day 3: Collection]
Cells are ready to collect for various assays - after transfection step.