Jan 23, 2023
Microscopy-based evaluation of Parkin translocation and mitophagy in FBXO7-/- cell lines
- 1Harvard Medical School
Protocol Citation: Felix Kraus 2023. Microscopy-based evaluation of Parkin translocation and mitophagy in FBXO7-/- cell lines. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gpjw28gzp/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: In development
We are still developing and optimizing this protocol
Created: October 11, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 71185
Keywords: ASAPCRN
Funders Acknowledgement:
ASAP
Grant ID: ASAP-000282
Abstract
Microscopy-based evaluation of Parkin translocation and mitophagy in FBXO7-/- cell lines
Seeding of HeLa
Seeding of HeLa
Wash HeLa cells expressing GFP-Parkin with 1x PBS
Add Trypsin to cells for 5 min and incubate at 37°C to dissociate cells from plastic well
Resuspend cells in 1 mL DMEM media
Count cells
Seed appropriate number of cells into 24-well glass bottom dish
Top up glass bottom dish with either 1 mL DMEM and place cells back into incubator
Induce mitophagy using Antimycin A / Oligomycin A for the desired time.
Staining
Staining
Aspirate DMEM and fix cells in 1 ml pre-warmed 4% PFA for 30 min.
Aspirate PFA solution and wash wells 3x with PBST (1x PBS, 0.02% Tween 20)
Permeabilize the cells by adding 0.2% Triton X-100 in PBS.
Remove the detergent solution by aspiration. Wash wells 3x with PBST (1x PBS, 0.02% Tween 20). Drain well.
Block cells for 10 min with 3% BSA – 1x PBS.
Remove BSA solution by aspiration. Wash wells 3x with PBST (1x PBS, 0.02% Tween 20). Drain well.
Incubate with primary antibodies in 3% BSA - 1x PBS for 3h at RT with gentle shaking. a. Anti-GFP (chicken) b. Anti-HSP60 (mouse)
Wash wells 3x with PBST (1x PBS, 0.02% Tween 20). Drain well.
Incubate with secondary antibodies in 3% BSA - 1x PBS for 45 min – 1h. a. Goat anti-chicken AlexaFlour 488 b. Goat anti-mouse AlexaFluor 647 c. DNA-SPY555
Wash wells 3x with PBST (1x PBS, 0.02% Tween 20). Drain well.
Wash wells 3x with PBST (1x PBS, 0.02% Tween 20). Drain well.
Exchange PBST with 1x PBS and keep cells at 4°C until imaging. Image within the next few days.
Fixed-cell microscopy
Fixed-cell microscopy
Mount glass bottom plate on Yokogawa CSU-W1 spinning disk confocal on a Nikon Eclipse Ti-E motorized microscope equipped with a Nikon Apochromat 60×/1.42 N.A oil-objective lens. Image signals of 488/568/647 fluorophores in sequential manner with a Nikon LUN-F XL solid state laser combiner ([laser line – laser power]: 488 - 80mW, 561 - 65mW, 640nm - 60mW]) using a Semrock Di01-T405/488/568/647 dichroic mirror. Fluorescence emissions were collected with 488 Chroma ET525/50m [488 nm], 568 Chroma ET605/52m [561 nm], 633 Chroma ET705/72m [640 nm] filters, respectively (Chroma Technologies) using NIS-Elements image acquisition software. Consistent laser intensity and exposure times must be maintained for all samples. Acquire 8 µm z-stacks for each image.
Image adequate number of cells per repeat in each condition.
Evaluation
Evaluation
Perform image quantification was in your tool of choice. Here we will use CellProfiler and segmentation pipelines can be found online (https://github.com/harperlaboratory/FBXO7).
Export results and plot in your tool of choice for graphing and statistical analysis.