Jul 31, 2023

Public workspaceHeLa culture, transfection, and labeling of Halo-fusion proteins

DOI
dx.doi.org/10.17504/protocols.io.yxmvmnb66g3p/v1
  • 1Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104;
  • 2Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815
OLIVIA HARDING
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DOI: dx.doi.org/10.17504/protocols.io.yxmvmnb66g3p/v1
Protocol Citation: OLIVIA HARDING, Erika L.F. Holzbaur 2023. HeLa culture, transfection, and labeling of Halo-fusion proteins. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvmnb66g3p/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 21, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 65012
Keywords: tissue culture, transfection, fluorescent ligands, ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson’s
Grant ID: Mechanisms of mitochondrial damage control by PINK1 and Parkin (ASAP-000350)
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.
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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.
Materials
Materials:

  • ReagentCountess slidesThermo Fisher ScientificCatalog #C10228
  • Reagent10 ml conical tubeCorningCatalog #CLS430055
  • Reagent1.5 mL capped tubesMillipore SigmaCatalog #EP022364120
  • Reagent35 mm glass-bottomed dishesMatTek CorporationCatalog #P35G-1.5-20-C

Reagents:

  • ReagentTrypsinThermo Fisher ScientificCatalog #R001100
  • ReagentTrypan blueThermo Fisher ScientificCatalog #T10282
  • ReagentDMEMCorningCatalog #10-017-CV
  • FBS (HyClone)
  • ReagentGlutaMAX glucose supplementGibco - Thermo FischerCatalog #35050061
  • ReagentOpti-MEMThermo Fisher ScientificCatalog #3198507
  • Plasmid DNA

  1. Untagged Parkin (subcloned from YFP-Parkin, a gift from R. Youle, NIH, Bethesda, MD)
  2. Mito-DsRed2 (kindly provided by. T. Schwartz, Harvard Medical School, Boston)
  3. Mito-sBFP2 (Wong and Holzbaur, PNAS, 2014)
  4. EGFP-NEM(kindly provided by E. Laplantine, Institut Pasteur, Paris),
  5. EGFP-NEMOD304N (generated by site-directed mutagenesis of EGFPNEMO),
  6. Halo-NEM(subcloned from EGFP-NEMO);
  7. Halo-OPTN (subcloned from EGFP-OPTN kindly provided from I. Dikic, Goethe University, Frankfurt, ta pHaloTag vector, Promega);
  8. mCherry constructs (vector, WT, ΔPB1, PB1AA, ΔUBA, and LIRAAAA, were kindly provided by S. Martens, University of Vienna, Austria, and TIRAAA was generated by site-directed mutagenesis of mCherry-p62WT)
  9. ReagentIKK2-EGFP (Plasmid #111195)addgeneCatalog #111195
  10. ReagentpCellFree_G03 TFAMaddgeneCatalog #67064
  11. ReagentmRFP-UbaddgeneCatalog #11935


Equipment:

Equipment
Countess II
NAME
Life Technologies
BRAND
AMQAX1000
SKU

  • mini centrifuge (Southern Labware, MLX-106)
  • Compound microscope








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 Temperature4 °C and warm to Temperature37 °C before use.

Day 1: Plating
Day 1: Plating
Follow plating protocol as described in dx.doi.org/10.17504/protocols.io.bt7wnrpe.
Day 2: Transfection
Day 2: Transfection
1d 18h 30m 4s
1d 18h 30m 4s
Examine cells by compound microscope Duration18:00:00 -Duration24:00:00 after plating to confirm 80-90% confluence.
Note
Note: If cells are not at 80-90% confluence, do not transfect. Wait until they reach 80-90%.

1d 18h
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

  • Amount200 µL Opti-mem
  • Amount0.2 µg mito-dsRed
  • Amount0.5 µg Parkin
  • Amount0.2 µg EGFP-NEMO
Note
Note: 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.
  • Amount0.5 µg Halo-OPTN

Tube 2 (Lipofectamine 2000)

  • Amount200 µL Opti-mem
  • Amount1.5 µL Lipofectamine 2000
Note
Note: 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 Duration00:05:00 -Duration00:10:00 at TemperatureRoom temperature .
15m
Incubation
Spin Duration00:00:02 in a mini centrifuge.
2s
Centrifigation
Add Tube 2 to Tube 1 and invert 8 times to mix.
Pipetting
Mix
Incubate Duration00:05:00 -Duration00:10:00 at TemperatureRoom temperature .
15m
Incubation
Spin Duration00:00:02 in a mini centrifuge.
2s
Centrifigation
Add entire volume (~ >400 uL) to the cells dropwise, distributing the drops mostly in the center of the dish (where the imaging window is).
Pipetting
Day 3: Labeling with fluorescent Halo ligands
Day 3: Labeling with fluorescent Halo ligands
30m
30m
Prepare Halo Dilution A by making a 1:200 dilution of stock Halo ligand in Culture Media.
Note
  • Will use 20 uL Dilution A per dish
  • Can freeze Dilution A at -20 for up to several months

Prepare working ligand solution by transferring Amount280 µL conditioned media from the dish where cells are plated to a 1.5 mL tube and adding Amount20 µL Halo Dilution A.
Pipetting
Transfer the rest of the conditioned media in the well (~2 mL) to a 10 mL conical tube and store in a Temperature37 °C water bath.
Pipetting
Gently drop Dilution A onto cells.
Incubate cells at Temperature37 °C , 5% CO2 for at least Duration00:15:00 .

Note
Note: this incubation step can be up to 2 hr, but a longer incubation introduces the risk of media evaporation

15m
Incubation
Remove the cells from the incubator and aspirate ligand solution with vacuum.
Wash cells gently with ~Amount200 µL conditioned media.
Wash
Aspirate wash media and repeat for a total of 2 washes.
Add Amount300 µL conditioned media.
Pipetting
Replace plated cells in incubator and rest for Duration00:15:00 .
15m
Incubation
Wash cells gently with ~Amount200 µL conditioned media.
Wash
Aspirate wash media and repeat for a total of 2 washes.
Cells are prepared for fixation or imaging.