HeLa culture, transfection, and labeling of Halo-fusion proteins
OLIVIA HARDING, Erika L.F. Holzbaur
Abstract
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.
Attachments
Steps
Day 1: Plating
Follow plating protocol as described in dx.doi.org/10.17504/protocols.io.bt7wnrpe.
Day 2: Transfection
Examine cells by compound microscope 18h 0m 0s-24h 0m 0s after plating to confirm 80-90% confluence.
For each dish, prepare the mixture of desired plasmids in 1.5 mL tubes.
For example, to characterize percent of mitochondria that recruit NEMO after depolarization, to Tube 1 (nucleic acids) add
200µLOpti-mem0.2µgmito-dsRed0.5µgParkin0.2µgEGFP-NEMO*NoteNote: NEMO over-expression must be kept to low levels in order to avoid activating cell response pathways. In our preliminary work, we established that 0.2 ug or less is ideal to transfect for a 35 mm dish. 0.5µgHalo-OPTN
Tube 2 (Lipofectamine 2000)
200µLOpti-mem1.5µLLipofectamine 2000NoteNote: The Lipofectamine 2000 volume needed is less than previously reported and less than recommended on the product datasheet. Using greater volumes of this reagent is toxic to cells.
Invert tubes 8 times to distribute the contents.
Incubate 0h 5m 0s-0h 10m 0s at Room temperature.
Spin 0h 0m 2s in a mini centrifuge.
Add Tube 2 to Tube 1 and invert 8 times to mix.
Incubate 0h 5m 0s-0h 10m 0s at Room temperature.
Spin 0h 0m 2s in a mini centrifuge.
Add entire volume (~ >400 uL) to the cells dropwise, distributing the drops mostly in the center of the dish (where the imaging window is).
Day 3: Labeling with fluorescent Halo ligands
Prepare Halo Dilution A by making a 1:200 dilution of stock Halo ligand in Culture Media.
Prepare working ligand solution by transferring 280µL conditioned media from the dish where cells are plated to a 1.5 mL tube and adding 20µL Halo Dilution A.
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.
Gently drop Dilution A onto cells.
Incubate cells at 37°C, 5% CO2 for at least 0h 15m 0s.
Remove the cells from the incubator and aspirate ligand solution with vacuum.
Wash cells gently with ~200µL conditioned media.
Aspirate wash media and repeat for a total of 2 washes.
Add 300µL conditioned media.
Replace plated cells in incubator and rest for 0h 15m 0s.
Wash cells gently with ~200µL conditioned media.
Aspirate wash media and repeat for a total of 2 washes.
Cells are prepared for fixation or imaging.
