TBK1 knockdown and rescue in Hela-M cells

OLIVIA HARDING, Olivia Harding, Erika L.F: Holzbaur

Published: 2021-09-01 DOI: 10.17504/protocols.io.bt7wnrpe

Abstract

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.

Before start

Cells are best transfected before passage 30. Higher passage number could result in lower transfection efficiency. Use cells between P5 and P25 for best results.* siRNA is easily degraded by RNAases that exist on everyday surfaces. Follow best practices for handling siRNA by suspending desiccated reagent in RNAase-free water, aliquoting into sterilized tubes, and using barrier pipet tips to handle aliquots.

Attachments

db5nbjnsp.pdf

Steps

Day 1: Plating

Trypsinize Hela-M cells by aspirating all media from a 10 cm dish of confluent cells, then dropping 0.75mL onto cells.

Incubate cells at 37°C, 5% for0h 5m 0s.

Resuspend detached cells and neutralize Trypsin with 1mL with 10% and 1% for a final volume of 1.75mL.

Transfer this volume to 10 mL conical tube.

Combine 10µL with 10µL in a 1.5 mL tube.

Drop 10µL onto a Countess slide and insert into the cell counter to calculate the concentration of cells in the resuspended solution.

Plate ~0.25 million HeLaM cells on 35mm imaging dish in 2mL.

Note

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.

Note

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 18h 0m 0s - 24h 0m 0s after plating to confirm 80-90% confluence.

Note

If cells are not at 80-90% confluence, do not transfect. Wait until they reach 80-90%.

For each dish, prepare the following two solutions in 1.5 mL tubes.

Tube 1 (nucleic acids): 200µL

- 0.5µL (stock at 1µg/µL)
- 0.25µL(“ “)
- 0.5µL(“ “)
- 4.8µL
- 4.8µL
  Note
  Invert stock solutions of each plasmid several times in order to ensure even distribution of plasmid.

Tube 2 (Lipofectamine 2000): 200µL

- 11.4µL
  Note
  The Lipofectamine 2000 volume is calculated by this equation:(X4)+(Y3.2), where X = ug plasmid DNA (in this protocol, X=1.25) and Y = # of 4.8 µL aliquots of siRNA(in this protocol, Y = 2)

10.

Invert tubes 8 times to distribute the contents, then:

11.

Incubate 0h 5m 0s -0h 10m 0s at Room temperature .

12.

Spin 0h 0m 2s in a minicentrifuge.

13.

Add Tube 2 to Tube 1 and invert 8 times to mix.

14.

Incubate 0h 5m 0s - 0h 10m 0s at 37Room temperature.

15.

Spin 0h 0m 2s in a minicentrifuge.