TITLE: Immunofluorescence of Galectin-3 Puncta after lysosomal damage with LLoMe    
AUTHORS: Vinay V. Eapen, Sharan Swarup, Melissa Hoyer, Harper   JW

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">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.</div></div>

[STEPS]
?. [Immunofluorescence of Galectin-3 Puncta after lysosomal damage with LLoMe]
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.
For HeLa cells, we use Dulbecco’s MEM (DMEM), high glucose (4500 mg/L), pyruvate (100 mg/L) supplemented with 10% fetal bovine serum.
?. [Immunofluorescence of Galectin-3 Puncta after lysosomal damage with LLoMe]
Treat the cells with  –  of LLoMe for .
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.
?. [Immunofluorescence of Galectin-3 Puncta after lysosomal damage with LLoMe]
Remove LLoMe containing media from the cells and replace with fresh media not containing LLoMe.
?. [Immunofluorescence of Galectin-3 Puncta after lysosomal damage with LLoMe]
After 10h, Wash the cells one time with phosphate buffered saline (PBS) and then fix with 4% paraformaldehyde in PBS for  at .
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?. [Immunofluorescence of Galectin-3 Puncta after lysosomal damage with LLoMe]
Remove 4% paraformaldehyde in PBS, wash the cells once with PBS, and solubilize cells with 0.1% triton-X in PBS for  at .
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?. [Immunofluorescence of Galectin-3 Puncta after lysosomal damage with LLoMe]
Block the cells are for  at  with sterile filtered blocking buffer (1% bovine serum albumin, 0.1% triton-X in PBS).
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?. [Immunofluorescence of Galectin-3 Puncta after lysosomal damage with LLoMe]
Add primary antibodies to blocking buffer at 1:300 and then spun down for  at . Remove the blocking buffer is completely and then apply the antibody in blocking buffer to the cells ( applied to the center of the well where the glass coverslip is attached) for  at .
Centrifuge: 10000 34
100 µl
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?. [Immunofluorescence of Galectin-3 Puncta after lysosomal damage with LLoMe]
Wash the cells 4 times with PBS ( for each wash).
?. [Immunofluorescence of Galectin-3 Puncta after lysosomal damage with LLoMe]
Add fluorescently conjugated secondary antibodies to blocking buffer at 1:300 and then spun down for  at . Remove the blocking buffer is completely and then apply the antibody in blocking buffer to the cells (100L applied to the center of the well where the glass coverslip is attached) for  at .
Centrifuge: 10000 34
0 Room temperature
?. [Immunofluorescence of Galectin-3 Puncta after lysosomal damage with LLoMe]
Wash the cells 4 times with PBS ( for each wash) and left in PBS.
?. [Immunofluorescence of Galectin-3 Puncta after lysosomal damage with LLoMe]
Image the cells at  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.
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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.
?. [Immunofluorescence of Galectin-3 Puncta after lysosomal damage with LLoMe]
Detect Galectin-3 puncta using CellProfiler with the same pipeline applied for each condition (see attached CellProfiler pipeline).
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.
?. [Immunofluorescence of Galectin-3 Puncta after lysosomal damage with LLoMe]
Each channel z series are brightness and adjust contrast equally and then convert to rgb for publication using FIJI software.