Fixation and imaging of HeLa cells after mitochondrial depolarization

OLIVIA HARDING; Erika L.F. Holzbaur

Aug 01, 2023

Fixation and imaging of HeLa cells after mitochondrial depolarization

DOI

https://dx.doi.org/10.17504/protocols.io.n92ldz6oxv5b/v1

OLIVIA HARDING^1,2,
Erika L.F. Holzbaur^1,2

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

Children's Hospital of Philadelphia

DOI: https://dx.doi.org/10.17504/protocols.io.n92ldz6oxv5b/v1

Protocol Citation: OLIVIA HARDING, Erika L.F. Holzbaur 2023. Fixation and imaging of HeLa cells after mitochondrial depolarization. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ldz6oxv5b/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 22, 2022

Last Modified: June 01, 2024

Protocol Integer ID: 65083

Keywords: Mitochondrial depolarization, Paraformaldehyde fixation, Confocal imaging, ASAPCRN, mitochondrial depolarization ectopic expression of p62, poor cell health due to p62, p62 mutation, tagged p62 mutation, ectopic expression of p62, toxic artifacts of p62 overexpression, depolarized mitochondria, mitochondrial depolarization, p62 overexpression, cell fixation protocol, p62 at low level, tagged p62, various endogenous atg8 protein, imaging of hela cell, p62, hela cell, cell health, cell, poor cell health, live cell

Funders Acknowledgements:

Aligning Science Across Parkinson’s

Grant ID: Mechanisms of mitochondrial damage control by PINK1 and Parkin (ASAP-000350)

Abstract

Ectopic expression of p62/SQSTM1 often induces puncta formation and poor cell health due to p62’s proclivity to multimerize and form filaments. We used the cell fixation protocol described here in order to over-express fluorescently tagged p62 at low levels in p62-/- cells and amplify the signal with immuno-labeling. By this method, we were able to express a variety of tagged p62 mutations to determine their effects on NEMO recruitment to depolarized mitochondria without introducing toxic artifacts of p62 overexpression. We also note the use of a modified fixation protocol to visualize various endogenous ATG8 proteins, which are also difficult to over-express in our system. Thus, fixation techniques are a critical complement to studies in live cells. 

Attachments

470-982.pdf

320KB

Guidelines

This protocol was adapted from a previous protocol for similar techniques  
      (see dx.doi.org/10.17504/protocols.io.bujsnune)
Here we use a primary antibody to a mitochondrial protein, HSP60, in order to identify mitochondria, instead of employing a genetically encoded fluorescent protein. While there are excellent mitochondrially-targeted fluorescent proteins, using anti-HSP60 allows us to demonstrate the versatility of the protocol to image both expressed, tagged proteins and immune-labeled structures.

Materials

Materials

1.5 mL capped tubesMerck MilliporeSigma (Sigma-Aldrich)Catalog #EP022364120  
 10 ml conical tubeCorningCatalog #CLS430055  
 Aluminum foil (Reynolds) 
Jewelers forceps Dumont No. 5Merck MilliporeSigma (Sigma-Aldrich)Catalog #F6521-1EA  
 Kimtech Science™ Kimwipes™ Delicate Task WipesKimberly-ClarkCatalog #34155  
PARAFILM® MMerck MilliporeSigma (Sigma-Aldrich)Catalog #P7793-1EA  
Tape
Corning® tissue-culture treated culture dishesMerck Millipore (EMD Millipore)Catalog #CLS430599-60EA  
Fisherbrand™ Premium Frosted Microscope Slides Superfrost marking areaFisher ScientificCatalog #12-544-2  
VectaShield PlusVector LaboratoriesCatalog #H-1000-10  
Nail polish or similar sealant



Reagents: 

Corning® 500 mL DMEM (Dulbecco’s Modified Eagle’s Medium)CorningCatalog #10-017-CV  
 FBS (HyClone)
 GlutaMAX&trade; SupplementThermo FisherCatalog #35050061  
Dimethyl sulfoxide (DMSO)Merck MilliporeSigma (Sigma-Aldrich)Catalog #D2650 
Ethanol 
Antimycin A from Streptomyces sp.Merck MilliporeSigma (Sigma-Aldrich)Catalog #A8674  and
Oligomycin AMerck MilliporeSigma (Sigma-Aldrich)Catalog #75351  
ParaformaldehydeMerck MilliporeSigma (Sigma-Aldrich)Catalog #158127  
Phosphate buffered saline (PBS)
Triton X-100 Merck MilliporeSigma (Sigma-Aldrich)Catalog #11332481001  
Fisher BioReagents™ Bovine Serum Albumin Fraction V Cold-ethanol PrecipitatedFisher ScientificCatalog # BP1605100  
Anti-HSP60 antibody produced in rabbitMerck MilliporeSigma (Sigma-Aldrich)Catalog #SAB4501464  
Anti-SQSTM1 / p62 antibody [2C11] - BSA and Azide free AbcamCatalog #ab56416  
Rabbit IgG (H L) Highly Cross-Adsorbed Secondary Antibody (A48254) in ICC/IFThermo Fisher ScientificCatalog #A48254  
Mouse IgG (H L) Highly Cross-Adsorbed Secondary Antibody (A-11030) in ICC/IFThermo Fisher ScientificCatalog #A-11030  


Equipment:

Cell incubator to maintain 37 °C   and 5% CO2 atmosphere
Vacuum apparatus
37 °C   water bath
Centrifuge, refrigerated
Confocal microscope with 60X objective associated software

Before start

The start point for this protocol is after cells grown on glass coverslips in a 12-well plate have been transfected with Parkin, EGFP-NEMO, HaloOPTN, and mCherry-p62 for 18- 24:00:00   and tagged with Halo ligand.
Prepare 45 millimolar (mM)   stock of Antimycin A by suspending 50 mg   solid AntA in 2 mL   ethanol.
 Prepare 10 millimolar (mM)   stock Oligomycin A by suspending 5 mg   solid OligA in 630 µL   DMSO
Prepare 4% PFA in PBS. Keep frozen at -20 °C  .
Note
Prepare fresh for day-of fixation or thaw directly before use.
Will use 1 mL   4% PFA per well.
 
Prepare Culture Media by making 10% FBS, 1% GlutaMAX solution in DMEM.
Prepare 0.5% Triton X-100 in PBS (Permeabilization buffer).
Note
Prepare 0.75 mL   Permeabilization buffer per well, store at 4 °C  .
Bring to Room temperature   before use. This will provide less shock to cells, better preserving fixed structures.
Do not use Triton for permeabilization if LC3- autophagosomes are the structure of interest, since Triton is too harsh. Use ice cold methanol if imaging LC3 structures



Prepare 0.2% Triton X-100/3% BSA in PBS (Blocking buffer).
Note
Prepare 0.75 mL   Blocking buffer per well, store at 4 °C  .
Bring to Room temperature   before use.


  Prepare a humidity chamber as previously described (dx.doi.org/10.17504/protocols.io.bujsnune)

AntA/OligA treatment

 
Prepare working AntA/OligA solution by transferring 0.5 mL   conditioned media to a 1.5 mL tube and adding 0.25 µL   10 millimolar (mM)   OligA and 2 mL   45 millimolar (mM)   AntA.
 

Gently drop working AntA/OligA solution onto cells.

 Incubate at 37 °C  , 5% CO2 for 00:55:00  - 01:05:00  .
Note
Our protocol calls for ~01:00:00   mitochondrial damage since that was our time course of interest. Users may vary the time course in order to examine earlier or later effects of global mitochondrial damage.
To carry out vehicle control experiment, use the equivalent amounts of ethanol and DMSO treatment. 

Fixation

25 minutes before AntA/OligA treatment is complete, warm 4% PFA and 1X PBS to 37 °C  .
 

Remove cells from incubator and aspirate media.
Note
When possible, keep cells covered with a sheet of aluminum foil, since fluorescent ligands are light-sensitive.

Quickly drop on 0.5 mL   warmed 1X PBS.

Aspirate PBS.

 Repeat warm PBS wash.

Drop on 1 mL   warmed 4% PFA.

 Incubate at 37 °C   for 00:10:00  .
Note
Warmed PBS and 4% PFA need not be sterile. Thus, if cells are no longer sterile, incubate in a non-sterile 37 °C   environment.

10m

Drop on 0.5 mL   warmed PBS.

Aspirate PBS.

Drop on 0.5 mL   warmed PBS.

Incubate covered at Room temperature   for 00:03:00  .
Note
Cells can be covered and stored at 4 °C   for up to several days after this step. If you choose to pause at this step, add at least 1.5 mL   PBS to wells to prevent evaporation.

Permeabilization

 
 Aspirate PBS.
 

Drop on 0.75 mL   Permeabilization buffer.

 Incubate covered at Room temperature   for 00:05:00  .
Note
If fixation protocol is used to examine autophagosomal structures, do not use buffer with Triton, as this will destroy the vesicles. Instead, use ice cold methanol permeabilization buffer and goat serum/BSA blocking buffer.

Blocking

 
 Add ~250 µL   Blocking buffer to each well.
 

Incubate covered at Room temperature   for 00:45:00  .
Note
Blocking step can be up to 01:00:00  .
Prepare primary antibody dilution during this step.

45m

Aspirate Blocking buffer.

Primary antibody

 
Prepare 200 µL   Primary antibody dilution per coverslip, with 1:250 antiHSP60 and 1:500 anti-p62 in Blocking buffer.
Note
Before use, spin primary antibody at top speed in a refrigerated centrifuge for 00:05:00  . Pipet from top of solution to ensure that no aggregates are present.
While mCherry-p62 was transfected in these cells, anti-p62 antibody boosts the signal.

Use sharp forceps to carefully lift the coverslip out of the well and dab excess Blocking buffer on a Kimwipe.

Place slip cell-side up on Parafilm in the humidity chamber.

 From the edge of the coverslip, pipet primary antibody dilution onto the cells.

Incubate covered at 4 °C   Overnight  .
Note
Twist two Kimwipes and wet with water so that they are more than damp but not dripping. Pack the wet Kimwipes into the edges of the humidity chamber in order to prevent evaporation of antibody
solution during the incubation.

18- 24 hours later, aspirate antibody dilution from the edge of the coverslip, gently lifting one side of the slip with forceps if necessary to allow buffer to slide off.

From the edge of the coverslip, pipet 100 µL   Room temperature   PBS onto the cells.

 Incubate covered at Room temperature   for 00:05:00  .

Aspirate PBS from the edge of the coverslip, gently lifting one side of the slip with forceps if necessary to allow buffer to slide off.

Repeat previous three steps for a total of four washes.

Wash (1/4):
From the edge of the coverslip, pipet 100 µL   Room temperature   PBS onto the cells.
Incubate covered at Room temperature   for 00:05:00  .
Aspirate PBS from the edge of the coverslip, gently lifting one side of the slip with forceps if necessary to allow buffer to slide off.

Wash (2/4):
From the edge of the coverslip, pipet 100 µL   Room temperature   PBS onto the cells.
Incubate covered at Room temperature   for 00:05:00  .
Aspirate PBS from the edge of the coverslip, gently lifting one side of the slip with forceps if necessary to allow buffer to slide off.

Wash (3/4):
From the edge of the coverslip, pipet 100 µL   Room temperature   PBS onto the cells.
Incubate covered at Room temperature   for 00:05:00  .
Aspirate PBS from the edge of the coverslip, gently lifting one side of the slip with forceps if necessary to allow buffer to slide off.

Wash (4/4):
From the edge of the coverslip, pipet 100 µL   Room temperature   PBS onto the cells.
Incubate covered at Room temperature   for 00:05:00  .
Aspirate PBS from the edge of the coverslip, gently lifting one side of the slip with forceps if necessary to allow buffer to slide off.

Secondary antibody

 
 Prepare secondary antibody solution by diluting goat anti-rabbit 405 and goat anti-mouse 1:200 in Blocking buffer.
Note
Prepare 125 µL   secondary solution for each coverslip.
Before use, spin primary antibody at top speed in a refrigerated centrifuge for 00:05:00  . Pipet from top of solution to  ensure that no aggregates are present.

From the edge of the coverslip, pipet secondary antibody dilution onto the cells.

 Incubate covered at Room temperature   for 00:45:00  .
Note
Secondary incubation can be up to 01:00:00  .
 Warm VectaShield to Room temperature   during this step.

45m

Aspirate antibody dilution from the edge of the coverslip, gently lifting one side of the slip with forceps if necessary to allow buffer to slide off.

From the edge of the coverslip, pipet 100 µL    Room temperature   PBS onto the cells.
 

Incubate covered at Room temperature   for 00:05:00  .

Aspirate PBS from the edge of the coverslip, gently lifting one side of the slip with forceps if necessary to allow buffer to slide off.

Repeat previous three steps for a total of four washes.

Wash (1/4)
From the edge of the coverslip, pipet 100 µL    Room temperature   PBS onto the cells.
Incubate covered at Room temperature   for 00:05:00  .
Aspirate PBS from the edge of the coverslip, gently lifting one side of the slip with forceps if necessary to allow buffer to slide off.

Wash (2/4)
From the edge of the coverslip, pipet 100 µL    Room temperature   PBS onto the cells.
Incubate covered at Room temperature   for 00:05:00  .
Aspirate PBS from the edge of the coverslip, gently lifting one side of the slip with forceps if necessary to allow buffer to slide off.

Wash (3/4)
From the edge of the coverslip, pipet 100 µL    Room temperature   PBS onto the cells.
Incubate covered at Room temperature   for 00:05:00  .
Aspirate PBS from the edge of the coverslip, gently lifting one side of the slip with forceps if necessary to allow buffer to slide off.

Wash (4/4)
From the edge of the coverslip, pipet 100 µL    Room temperature   PBS onto the cells.
Incubate covered at Room temperature   for 00:05:00  .
Aspirate PBS from the edge of the coverslip, gently lifting one side of the slip with forceps if necessary to allow buffer to slide off.

Mounting

 
Pipet 12.5 µL   Room temperature   VectaShield onto a microscope slide.
 

Pick up coverslip with forceps and dab excess PBS onto a Kimwipe.

Lay coverslip cell-side down onto VectaShield drop.

Seal edges of coverslip with nail polish.

Lay flat until set.
Note
Store slides at 4 °C  .

Imaging

Using 60X objective and RFP epifluorescence, find the focal plane of fixed cells by looking for dsRed2-labeled mitochondria.
Note
We find that 60X is sufficient magnification to collect several cells in each field of view with enough resolution to perform analysis and quantification. Other magnifications may be appropriate for various applications.

Configure 405, 488, 561, and 647 lasers and accompanying exposure times to maximize dynamic range of each signal.
Note
Since the cells are fixed, there is less downside to photo bleaching the samples.

Configure collection parameters to record a >2 um Z stack of images.
Note
On our system, 0.15 um is the minimum step size, thus 21 sections is sufficient.

Collect images from fields of view with multiple cells exhibiting all visible tags. Collect volume from the 50th to 75th percentile of the cell (see Figure 1). If mitochondria are depolarized, can use OPTN signal (recruitment to
fragmented mitochondria) to determine whether Parkin is expressed.

Figure 1. Schematic of HeLa cell volume. Dotted lines indicate target volume to image.

Collect images in order to record 10-20 cells per condition, depending on the quality of the transfection and cells. Collect images from diverse areas of the coverslip.