TITLE: HeLa culture, transfection, and labeling of Halo-fusion proteins
AUTHORS: OLIVIA HARDING, Erika L.F. Holzbaur

[DESCRIPTION]
High-throughput, predictable systems that are easily modulated are ideal for the study of cell biology. Here we developed a protocol to investigate the role of the Nuclear Factor kappa-B Effector Molecule (NEMO) in Parkin-dependent mitochondrial clearance. Transient transfection of fluorescent constructs allowed us to visualize subcellular structures and dynamics while maintaining flexibility in a consistent model system. The EGFP-NEMO plasmid was repeatedly employed to study NEMO interactions during mitophagy, and we were also able to edit the construct to create both a NEMO point mutation and a Halo-tagged NEMO construct, which we readily expressed in HeLa cells. Halo-fusion constructs, including NEMO and OPTN used in our study, allowed us to visualize the exogenously expressed proteins conjugated to chemical ligands in a variety of colors. This and the accompanying protocols were critical to our characterization of NEMO’s involvement in mitophagy.

[BEFORE_START]
HeLa-M cells are best transfected before passage 30; KO cells are best utilized before passage 15 and may grow slower than WT cells.
Prepare Culture Media by making a 10% FBS, 1% GlutaMAX solution in DMEM. Store at 4 °C and warm to 37 °C before use.


[GUIDELINES]
This protocol was adapted from a previous protocol for similar techniques (see dx.doi.org/10.17504/protocols.io.bt7wnrpe).
In order to investigate Parkin-dependent mitophagy, we over-express Parkin, which is not endogenously expressed in HeLa cells. We employ an untagged Parkin construct, however there are many permutations of fluorescently conjugated proteins that could be used to study this process.
We use HeLa-M cells, HeLa OPTN-/- cells, and HeLa p62-/- cells in the study corresponding to this protocol.

[STEPS]
SECTION: Day 1: Plating
1. Follow plating protocol as described in dx.doi.org/10.17504/protocols.io.bt7wnrpe.
SECTION: Day 2: Transfection
2. Examine cells by compound microscope 1080 min-1440 min after plating to confirm 80-90% confluence.
 

SECTION: Day 2: Transfection
3. For each dish, prepare the mixture of desired plasmids in 1.5 mL tubes.
SECTION: Day 2: Transfection
3.1. For example, to characterize percent of mitochondria that recruit NEMO after depolarization, to Tube 1 (nucleic acids) add

200 µL Opti-mem
0.2 µg mito-dsRed
0.5 µg Parkin
0.2 µg EGFP-NEMO
 
0.5 µg Halo-OPTN

SECTION: Day 2: Transfection
3.2. Tube 2 (Lipofectamine 2000)

200 µL Opti-mem
1.5 µL Lipofectamine 2000
 

SECTION: Day 2: Transfection
4. Invert tubes 8 times to distribute the contents.
SECTION: Day 2: Transfection
5. Incubate 5 min-10 min at Room temperature.
SECTION: Day 2: Transfection
6. Spin 2 s in a mini centrifuge.
SECTION: Day 2: Transfection
7. Add Tube 2 to Tube 1 and invert 8 times to mix.
SECTION: Day 2: Transfection
8. Incubate 5 min-10 min at Room temperature.
SECTION: Day 2: Transfection
9. Spin 2 s in a mini centrifuge.
SECTION: Day 2: Transfection
10. Add entire volume (~ >400 uL) to the cells dropwise, distributing the drops mostly in the center of the dish (where the imaging window is).
SECTION: Day 3: Labeling with fluorescent Halo ligands
11. Prepare Halo Dilution A by making a 1:200 dilution of stock Halo ligand in Culture Media.
 

SECTION: Day 3: Labeling with fluorescent Halo ligands
12. Prepare working ligand solution by transferring 280 µL conditioned media from the dish where cells are plated to a 1.5 mL tube and adding 20 µL Halo Dilution A.
SECTION: Day 3: Labeling with fluorescent Halo ligands
13. Transfer the rest of the conditioned media in the well (~2 mL) to a 10 mL conical tube and store in a 37 °C water bath.
SECTION: Day 3: Labeling with fluorescent Halo ligands
14. Gently drop Dilution A onto cells.
SECTION: Day 3: Labeling with fluorescent Halo ligands
15. Incubate cells at 37 °C, 5% CO2 for at least 15 min.

 

SECTION: Day 3: Labeling with fluorescent Halo ligands
16. Remove the cells from the incubator and aspirate ligand solution with vacuum.
SECTION: Day 3: Labeling with fluorescent Halo ligands
17. Wash cells gently with ~200 µL conditioned media.
SECTION: Day 3: Labeling with fluorescent Halo ligands
18. Aspirate wash media and repeat for a total of 2 washes.
SECTION: Day 3: Labeling with fluorescent Halo ligands
19. Add 300 µL conditioned media.
SECTION: Day 3: Labeling with fluorescent Halo ligands
20. Replace plated cells in incubator and rest for 15 min.
SECTION: Day 3: Labeling with fluorescent Halo ligands
21. Wash cells gently with ~200 µL conditioned media.
SECTION: Day 3: Labeling with fluorescent Halo ligands
22. Aspirate wash media and repeat for a total of 2 washes.
SECTION: Day 3: Labeling with fluorescent Halo ligands
23. Cells are prepared for fixation or imaging.