Oct 23, 2024
live and fixed cell fluorescence imaging of agDD-GFP aggregation and NRF1 activation
Forked from a private protocol
- Harper JW1,
- Kelsey Hickey1
- 1Department of Cell Biology, Harvard Medical School
Protocol Citation: Harper JW, Kelsey Hickey 2024. live and fixed cell fluorescence imaging of agDD-GFP aggregation and NRF1 activation. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl823e7l2w/v1
Manuscript citation:
https://www.biorxiv.org/content/10.1101/2022.12.06.519342v1
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: October 17, 2024
Last Modified: October 23, 2024
Protocol Integer ID: 110252
Keywords: ASAPCRN, protein aggregates
Funders Acknowledgement:
ASAP
Grant ID: 000282
Abstract
This protocol describes methods for visualization of agDD-GFP aggregates and NRF1 by fluorescence imaging and immunofluorescence, respectively.
Materials
HeLa-dCas9 cells (Ref 1 and 2) expressing agDD-GFP (Addgene #78289) as described (Ref 3)
PBS; Phosphate buffered saline: ThermoFisher (#14040133)
Dulbecco’s MEM (DMEM), high glucose, pyruvate (Gibco / Invitrogen, 11995)
18 or 22 mm-glass coverslips (No. 1.5, 22x22 mm glass diameter, VWR 48366-227)
FluoroBrite DMEM (Thermo Fisher Scientific A, 1896701)
NRF1/NFE2L1 (1:1000; abcam, ab238154)
mounting media (Vectashield H-1000)
AlexaFluor 594 Goat anti-Rabbit IgG (Thermo Fisher Catalog # A-11012)
Hoechst-33342 dye (Thermo Fisher #62249)
Cell Culture
Cell Culture
HeLa-dCas9 cells (from Ref 1 and 2) were grown in complete DMEM (Dulbecco’ Modifies Eagles Medium (DMEM) with 10% fetal bovine serum and optional 1% penicillin-streptomycin), with the addition of shield-1 (S1) ligand at a final concentration of 1 μm (MedChem Express, cat # HY-112210) to keep agDD-GFP in a folded form and maintained in a 5% CO2 incubator at 37oC. Cells were maintained at <80% confluency throughout the course of experiments.
Imaging of agDD-GFP aggregation in fixed cells
Imaging of agDD-GFP aggregation in fixed cells
Cells in the presence of S1 were plated onto 18 or 22 mm-glass coverslips (No. 1.5, 22x22 mm glass diameter, VWR 48366-227) for imaging. S1 washout was performed as described in Ref 3 using 5 μm FKBP12-F36V in the culture media. The choice of time for the washout will depend on the goals of the experiment but 10 min to 6 h represent the extremes of the time range, typically.
Cells were then fixed using 4% PFA for 10 min at room temperature.
Coverslips were then washed 3 times with DPBS + 0.02% tween-20, with the final wash done in the presence of Hoechst33342 dye to mark nuclei, and mounted onto glass slides using mounting media (Vectashield H-1000) and sealed with nail polish.
The cells were imaged by confocal microscopy using a Yokogawa CSU-W1 spinning disk confocal on a Nikon Ti motorized microscope equipped with a Nikon Plan Apo 100x/1.40 N.A objective lens, and Hamamatsu ORCA-Fusion BT CMOS camera. For the analysis, the equal gamma, brightness, and contrast are applied for each image using FiJi software (FiJi ImageJ V.2.0.0 https://imagej.net/Fiji).
Imaging of NRF1 in fixed cells
Imaging of NRF1 in fixed cells
Cells in the presence of S1 were plated onto 18 or 22 mm-glass coverslips (No. 1.5, 22x22 mm glass diameter, VWR 48366-227) for imaging. S1 washout was performed as described in Ref 3 using 5 μm FKBP12-F36V in the culture media. The choice of time for the washout will depend on the goals of the experiment but 10 min to 6 h represent the extremes of the time range, typically.
Cells were then fixed using 4% PFA followed by permeabilization with 0.5% Triton-X100.
Cells were blocked in 3% BSA for 30 minutes, followed by incubation in primary antibodies (e.g. anti-NRF1; 1:200) for 1 hour at room temperature. Cells were washed 3 times with PBS + 0.02% tween-20, followed by incubation in secondary (alexafluor conjugated secondary antibodies such as AlexaFluor 647 Goat anti-Rabbit IgG) for 1 hour at room temperature.
Coverslips were then washed 3 times with DPBS + 0.02% tween-20 and mounted onto glass slides using mounting media (Vectashield H-1000) and sealed with nail polish.
The cells were imaged by confocal microscopy. We employ a Yokogawa CSU-W1 spinning disk confocal on a Nikon Ti motorized microscope equipped with a Nikon Plan Apo 100x/1.40 N.A objective lens, and Hamamatsu ORCA-Fusion BT CMOS camera. For the analysis, the equal gamma, brightness, and contrast are applied for each image using FiJi software (FiJi ImageJ V.2.0.0 https://imagej.net/Fiji)
Imaging of agDD-GFP aggregation in live cells
Imaging of agDD-GFP aggregation in live cells
Cells in the presence of S1 were plated onto glass glass bottom dishes for imaging. S1 washout was performed as described in Ref 3 using 5 μm FKBP12-F36V in the culture media.
Before imaging, S1 washout is started to begin aggregation in PhenolRed free DMEM. Then
cells are imaged using a Yokogawa CSU-W1 spinning disk confocal on a Nikon Ti motorized microscope equipped with a Nikon Plan Apo 100x/1.40 N.A objective lens, and Hamamatsu ORCA-Fusion BT CMOS camera, and a live cell chamber with temperature and carbon dioxide control. For the analysis, the equal gamma, brightness, and contrast were applied for each image using FiJi software (FiJi ImageJ V.2.0.0 https://imagej.net/Fiji).
Protocol references
1. M. Jost et al., Combined CRISPRi/a-Based Chemical Genetic Screens Reveal that Rigosertib Is a
Microtubule-Destabilizing Agent. Mol Cell68, 210-223 e216 (2017).
2. L. A. Gilbert et al., CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell 154, 442-451 (2013).
3. Y. Miyazaki et al., A method to rapidly create protein aggregates in living cells. Nat Commun 7, 11689 (2016).