Sep 15, 2021
Version 2
Immunofluorescence of Galectin-3 Puncta after lysosomal damage with LLoMe V.2
- Vinay V. Eapen1,
- Sharan Swarup1,
- Melissa Hoyer1,
- Harper JW2
- 1dx.doi.org/10.17504/protocols.io.bxgypjxw;
- 2wade_harper@hms.harvard.edu
External link: https://doi.org/10.7554/eLife.72328
Protocol Citation: Vinay V. Eapen, Sharan Swarup, Melissa Hoyer, Harper JW 2021. Immunofluorescence of Galectin-3 Puncta after lysosomal damage with LLoMe . protocols.io https://dx.doi.org/10.17504/protocols.io.bx8pprvnVersion created by Harper JW
Manuscript citation:
Eapen VV, Swarup S, Hoyer MJ, Paulo JA, Harper JW, Quantitative proteomics reveals the selectivity of ubiquitin-binding autophagy receptors in the turnover of damaged lysosomes by lysophagy. eLife doi: 10.7554/eLife.72328
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: September 15, 2021
Last Modified: June 01, 2024
Protocol Integer ID: 53231
Keywords: Immunofluorescence, Galectin-3, Lysosomal damage, LLoMe, ASAPCRN
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Abstract
Lysophagy-the selective elimination of damaged lysosomes by the autophagy pathway-is a critical housekeeping mechanism in cells. This pathway surveils lysosomes and selectively demarcates terminally damaged lysosomes for elimination. Among the most upstream signaling proteins in this pathway are the glycan binding proteins-Galectins-which recognize N and O linked glycan chains on the luminal side of transmembrane lysosomal proteins. These glycosyl modifications are only accessible to galectin proteins upon extensive lysosomal membrane rupture and serve as a sensitive measure of lysosomal damage and eventual clearance by selective autophagy. Indeed, prior work has shown that immunofluorescence of Galectin-3 serves as a convenient proxy for lysophagic flux in tissue culture cells (Aits et al., 2015; Maejima et al., 2013). Here we describe our method for monitoring galectin-3 puncta clearance as a proxy for turnover of damaged lysosomes via immunofluorescence and confocal imaging.
Materials
Materials:
| A | B | C | |
| REAGENT or RESOURCE | SOURCE | IDENTIFIER | |
| Chemicals | |||
| LLoMe (L-Leucyl-L-Leucine methyl ester (hydrochloride)) | Cayman Chemical | 16008 | |
| Dulbecco’s MEM (DMEM), high glucose, pyruvate | GIBCO / Invitrogen | 11995 | |
| Phosphate Buffered Saline 1X | Corning | 21-031-CV | |
| Fetal Bovine Serum | Fisher | SH3008003 | |
| Bovine Serum Albumin | Gold biotechnology | A-420-250 | |
| paraformaldehyde | Electron Microscopy Sciences | 15710 | |
| Triton-X | Sigma | T8787 | |
| Antibodies | |||
| LAMP1 (D401S) Mouse mAb | Cell Signaling Technology | 15665S | |
| Galectin-3/ LGALS3 (M3/38) Rat mAb | Santa-Cruz | sc-23938 | |
| Software | |||
| Cell Profiler | CellProfiler v4.0.6 | https://cellprofiler.org/ | |
| Fiji | ImageJ V.2.0.0 | https://imagej.net/software/fiji/ | |
| Metamorph | Metamorph v | https://www.moleculardevices.com/products/cellular-imaging-systems/acquisition-and-analysis-software/metamorph-microscopy#gref |
Chemicals:
L-Leucyl-L-Leucine methyl ester (hydrochloride)Cayman Chemical CompanyCatalog #16008
DMEM, high glucose, pyruvateThermo FisherCatalog #11995065
Phosphate Buffered Saline (PBS)CorningCatalog #MT21-031-CV
ParaformaldehydeElectron Microscopy SciencesCatalog #15710
Triton X-100Sigma AldrichCatalog #T8787
Antibodies:
LAMP1 (D4O1S) Mouse mAb #15665Cell Signaling TechnologyCatalog #15665S
Anti-galectin-3 Antibody (M3/38): sc-23938Santa Cruz BiotechnologyCatalog #sc-23938
Immunofluorescence of Galectin-3 Puncta after lysosomal damage with LLoMe
Immunofluorescence of Galectin-3 Puncta after lysosomal damage with LLoMe
4h 12m
4h 12m
Plate the cells (selected by investigator) into 12 well glass bottom dishes (No. 1.5, 14 mm glass diameter, MatTek) are grown to 50-70% confluency in media.
Note
For HeLa cells, we use Dulbecco’s MEM (DMEM), high glucose (4500 mg/L), pyruvate (100 mg/L) supplemented with 10% fetal bovine serum.
Treat the cells with 500 micromolar (µM) – 1 millimolar (mM) of LLoMe for 01:00:00 .
Note
Note: The exact dosage varies with cell line and should be determined empirically depending on the line used. This dose range has been tested extensively in Hela cells, and routinely generated lysophagic flux.
1h
Remove LLoMe containing media from the cells and replace with fresh media not containing LLoMe.
After 10h, Wash the cells one time with phosphate buffered saline (PBS) and then fix with 4% paraformaldehyde in PBS for 00:15:00 at Room temperature .
15m
Remove 4% paraformaldehyde in PBS, wash the cells once with PBS, and solubilize cells with 0.1% triton-X in PBS for 00:15:00 at Room temperature .
15m
Block the cells are for 00:30:00 at Room temperature with sterile filtered blocking buffer (1% bovine serum albumin, 0.1% triton-X in PBS).
30m
Add primary antibodies to blocking buffer at 1:300 and then spun down for 00:01:00 at 10000 x g . Remove the blocking buffer is completely and then apply the antibody in blocking buffer to the cells (100 µL applied to the center of the well where the glass coverslip is attached) for 01:00:00 at Room temperature .
1h 1m
Wash the cells 4 times with PBS (00:05:00 for each wash).
5m
Add fluorescently conjugated secondary antibodies to blocking buffer at 1:300 and then spun down for 00:01:00 at 10000 x g . Remove the blocking buffer is completely and then apply the antibody in blocking buffer to the cells (100L applied to the center of the well where the glass coverslip is attached) for 01:00:00 at Room temperature .
1h 1m
Wash the cells 4 times with PBS (00:05:00 for each wash) and left in PBS.
5m
Image the cells at Room temperature using a Yokogawa CSU-X1 spinning disk confocal on a Nikon Ti-E inverted microscope at the Nikon Imaging Center in Harvard Medical School. Use Nikon Perfect Focus System to maintain cell focus over time. Equip the microscope with a Nikon Plan Apo 40x/1.30 N.A or 100x/1.40 N.A objective lens.
Note
445nm (75mW), 488nm (100mW), 561nm (100mW) & 642nm (100mW) laser lines are controlled by AOTF. All images are collected with a Hamamatsu ORCA-ER cooled CCD camera (6.45 µm2 photodiode) with MetaMorph image acquisition software.
Detect Galectin-3 puncta using CellProfiler with the same pipeline applied for each condition (see attached CellProfiler pipeline).
Note
Each cell area is first defined using a “identify primary objects” module that included objects 200 to 1000 pixels units, and each puncta is marked using a “identify primary objects” module that included objects 2 to 20 pixels units both with an optimized “robust background” threshold. Each cell for each condition is thresholded in the same way with a consistent pipeline. Object size and shape is measured, and each punctum is related to its respective cell to yield a puncta per cell readout. Each channel z series are brightness and contrast adjusted equally and then converted to rgb for publication using FIJI software.
Each channel z series are brightness and adjust contrast equally and then convert to rgb for publication using FIJI software.