TITLE: Fixation of HeLa-M cells expressing Halo and SNAP fusion proteins conjugated to ligands
AUTHORS: OLIVIA  HARDING, Erika L. F. Holzbaur

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Mitophagy is a tightly regulated mechanism in which components are sequentially recruited to a damaged mitochondrion in order to clear it by lysosomal degradation. Defects in mitophagy are thought to contribute to multiple human diseases, including neurodegenerative diseases; thus, further study of the pathway is imperative. Many of the molecular steps that comprise mitophagy were first investigated in the HeLa culture system before their translation to specific cell types. Here we developed a protocol for conjugating multiple mitophagy components exogenously expressed in HeLa cells to commercially available fluorescent ligands with the Halo (Promega) and SNAP (New England Biolabs) systems. We precisely control the time course of our investigation by fixing cells with paraformaldehyde after timed incubation with a mitochondrial poison that induces global mitophagy. After fixation, cells can be imaged for a variety of assays and quantifications.</div></div>

[STEPS]
?. [Before start]
The start point for this protocol is after cells grown on a glass coverslip in a 12- well plate have been transfected with YFP-Parkin, Halo-OPTN, and SNAP-TBK1 for - .
?. [Before start]
Prepare  in PBS. Keep frozen at  .
[PFA]
-20 °C
Prepare fresh for day-of fixation or thaw directly before use.
Will use   per well.
?. [Before start]
Prepare culture media by making a  ,  solution in DMEM, place in  water bath to warm.
[FBS]
[GlutaMAX]
37 °C
Prepare ~  per well.
?. [Before start]
Prepare Halo Dilution A by making a 1:200 dilution of stock Halo ligand in DMEM/FBS/GlutaMAX.
Will use   per well.
?. [Before start]
Prepare  in DMSO. This stock can be stored at  . Thaw at  when needed.
[CCCP stock]
-20 °C
0 Room temperature
Will use   per well.
?. [Before start]
Prepare  in PBS (Permeabilization buffer).
[Triton X-100]
Prepare   per well, store at  .
Bring to   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.
?. [Before start]
Prepare  in PBS (Blocking buffer).
[Triton X-100/3% BSA]
Prepare   per well, store at  .
Bring to    before use.
?. [Before start]
Prepare a humidity chamber by covering a 15-cm cell culture dish with aluminum foil. Cut an area of Parafilm to cover the inside of the dish, and tape each side. (See Figure 1)
?. [HaloTag]
Prepare working ligand solution by transferring  from the well where cells are plated to a 1.5 mL tube and adding .
[conditioned media]
[Halo Dilution A]
?. [HaloTag]
Transfer the rest of the conditioned media in the well (~2 mL) to a 10 mL conical tube and store in a   water bath.
37 °C
?. [HaloTag]
Gently drop working ligand solution onto cells.
?. [HaloTag]
Incubate cells at  ,  for at least .
37 °C
[CO2]
This incubation step can be up to  , but a longer incubation introduces the risk of media evaporation.
?. [HaloTag]
Remove the cells from the incubator and aspirate ligand solution with vacuum.
?. [HaloTag]
Wash cells by gently dropping .
[DMEM/FBS/GlutaMAX]
?. [HaloTag]
Aspirate wash media and repeat for a total of 2 washes.
?. [HaloTag]
Drop on .
[conditioned media]
?. [HaloTag]
Replace plated cells in incubator and rest for .
During this step, prepare working SNAP ligand solution.
?. [HaloTag]
Wash cells by gently dropping .
[DMEM/FBS/GlutaMAX]
?. [HaloTag]
Aspirate wash media and repeat for a total of 2 washes.
?. [SNAP Tag]
Prepare working SNAP ligand solution by transferring  from the conical tube to a 1.5 mL tube and adding .
[conditioned media]
[SNAP-Cell 647-SiR]
?. [SNAP Tag]
Gently drop working ligand solution onto cells.
?. [SNAP Tag]
Incubate cells at ,  for at least .
37 °C
[CO2]
This incubation step can be up to  , but a longer incubation introduces the risk of media evaporation.
?. [SNAP Tag]
Remove the cells from the incubator and aspirate ligand solution with vacuum.
?. [SNAP Tag]
Wash cells by gently dropping .
[DMEM/FBS/GlutaMAX]
?. [SNAP Tag]
Aspirate wash media and repeat for a total of 2 washes.
?. [SNAP Tag]
Drop on .
[conditioned media]
?. [SNAP Tag]
Replace plated cells in incubator and rest for .
During this step, prepare working CCCP solution
?. [SNAP Tag]
Wash cells by gently dropping .
[DMEM/FBS/GlutaMAX]
?. [SNAP Tag]
Aspirate wash media and repeat for a total of 2 washes.
?. [CCCP treatment]
Prepare working CCCP solution by transferring  from the conical tube to a 1.5 mL tube and adding .
[conditioned media]
[20 mM CCCP stock]
?. [CCCP treatment]
Gently drop working CCCP solution onto cells.
?. [CCCP treatment]
Incubate at ,  for .
37 °C
[CO2]
Our protocol calls for 1.5 hours mitochondrial damage since that was our timecourse of interest. Users may vary the timecourse in order to examine earlier or later effects of global mitochondrial damage.
?. [Fixation]
before CCCP treatment is finished, warm  to .
[PFA and 1X PBS]
37 °C
?. [Fixation]
Remove cells from incubator and aspirate media.
When possible, keep cells covered with a sheet of aluminum foil, since fluorescent ligands are light-sensitive.
?. [Fixation]
Quickly drop on .
[warmed 1X PBS]
?. [Fixation]
Aspirate PBS.
?. [Fixation]
Repeat warm PBS wash.
?. [Fixation]
Drop on .
[warmed 4% PFA]
?. [Fixation]
Incubate at  for .
37 °C
Warmed PBS and 4% PFA need not be sterile. Thus, if cells are no longer sterile, incubate in a non-sterile   environment.
?. [Fixation]
Drop on .
[warmed PBS]
?. [Fixation]
Aspirate PBS.
?. [Fixation]
Drop on .
[warmed PBS]
?. [Fixation]
Incubate covered at   for .
0 Room temperature
Cells can be covered and stored at   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.
?. [Permeabilization]
Drop on .
[Permeabilization buffer]
?. [Permeabilization]
Incubate covered at  for .
0 Room temperature
?. [Blocking]
Use sharp forceps to carefully lift the coverslip out of the well and dab excess Permeabilization buffer on a Kimwipe.
?. [Blocking]
Place slip cell-side up on Parafilm in the humidity chamber.
?. [Blocking]
From the edge of the coverslip, pipet  onto the cells.
[Blocking buffer]
?. [Blocking]
Incubate covered at  for .
0 Room temperature
Blocking step can be up to  .
Prepare primary antibody dilution during this step.
?. [Blocking]
Aspirate Blocking buffer from the edge of the coverslip, gently lifting one side of the slip with forceps if necessary to allow buffer to slide off.
?. [Primary antibody]
Prepare  at 1:125 in Blocking buffer for each coverslip.
[anti-HSP60]
Before use, spin primary antibody at top speed in a refrigerated centrifuge for  . Pipet from top of solution to ensure that no aggregates are present.
?. [Primary antibody]
From the edge of the coverslip, pipet primary antibody dilution onto the cells.
?. [Primary antibody]
Incubate covered at .
4 °C
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.
?. [Primary antibody]
-  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.
?. [Primary antibody]
From the edge of the coverslip, pipet  onto the cells.
[room temperature PBS]
?. [Primary antibody]
Incubate covered at   for .
0 Room temperature
?. [Primary antibody]
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.
?. [Primary antibody]
Repeat previous three steps for a total of four washes.
?. [Secondary antibody]
Prepare secondary antibody solution by diluting goat anti-rabbit 405 1:200 in Blocking buffer.
Prepare   for each coverslip.
Before use, spin primary antibody at top speed in a refrigerated centrifuge for  . Pipet from top of solution to ensure that no aggregates are present.
?. [Secondary antibody]
From the edge of the coverslip, pipet secondary antibody dilution onto the cells.
?. [Secondary antibody]
Incubate covered at   for .
0 Room temperature
Secondary incubation can be up to  .
Warm VectaShield to   during this step.
?. [Secondary antibody]
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.
?. [Secondary antibody]
From the edge of the coverslip, pipet  onto the cells.
[room temperature PBS]
?. [Secondary antibody]
Incubate covered at   for .
0 Room temperature
?. [Secondary antibody]
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]
Repeat previous three steps for a total of four washes.
?. [Mounting]
Pipet  onto a microscope slide.
[room temperature VectaShield]
?. [Mounting]
Pick up coverslip with forceps and dab excess PBS onto a Kimwipe.
?. [Mounting]
Lay coverslip cell-side down onto VectaShield drop.
?. [Mounting]
Seal edges of coverslip with nail polish.
?. [Mounting]
Lay flat until set.
?. [Mounting]
Samples can now be imaged.
Store slides at  .