TITLE: Immunofluorescence of autophagic cargo receptors and p-TBK1 at LAMP1 lysosomes during lysophagy
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 a method for monitoring protein recruitment to damaged lysosomes via immunofluorescence and confocal imaging.</div></div>

[STEPS]
?. [Immunofluorescence of autophagic cargo receptors and p-TBK1 at LAMP1 lysosomes during lysophagy]
Plate the cells (to be selected by the 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 autophagic cargo receptors and p-TBK1 at LAMP1 lysosomes during lysophagy]
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 autophagic cargo receptors and p-TBK1 at LAMP1 lysosomes during lysophagy]
Remove the LLoMe containing media from the cells and replace with fresh media not containing LLoMe.
?. [Immunofluorescence of autophagic cargo receptors and p-TBK1 at LAMP1 lysosomes during lysophagy]
After the indicated washout timepoint (4h for optimal receptor recruitment), wash the cells one time with phosphate buffered saline (PBS) and then fix with 4% paraformaldehyde in PBS for  at .
0 Room temperature
?. [Immunofluorescence of autophagic cargo receptors and p-TBK1 at LAMP1 lysosomes during lysophagy]
Remove 4% paraformaldehyde in PBS, wash the cells once with PBS, and then solubilize cells with 0.1% triton-X in PBS for  at .
0 Room temperature
?. [Immunofluorescence of autophagic cargo receptors and p-TBK1 at LAMP1 lysosomes during lysophagy]
Block the cells for  at  with sterile filtered blocking buffer (1% bovine serum albumin, 0.1% triton-X in PBS).
0 Room temperature
?. [Immunofluorescence of autophagic cargo receptors and p-TBK1 at LAMP1 lysosomes during lysophagy]
Add primary antibodies for relevant cargo adaptors or TBK1 to blocking buffer at 1:300 and then spun down for  at . Remove the blocking buffer 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
0 Room temperature
?. [Immunofluorescence of autophagic cargo receptors and p-TBK1 at LAMP1 lysosomes during lysophagy]
Wash the cells 4 times with PBS (5min for each wash).
?. [Immunofluorescence of autophagic cargo receptors and p-TBK1 at LAMP1 lysosomes during lysophagy]
Add fluorescently conjugated secondary antibodies to blocking buffer at 1:300 and then spun down for  at . Remove the blocking buffer 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
0 Room temperature
?. [Immunofluorescence of autophagic cargo receptors and p-TBK1 at LAMP1 lysosomes during lysophagy]
Wash the cells 4 times with PBS ( for each wash) and left in PBS.
?. [Immunofluorescence of autophagic cargo receptors and p-TBK1 at LAMP1 lysosomes during lysophagy]
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 the 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.
0 Room temperature
445nm (75mW), 488nm (100mW), 561nm (100mW) & 642nm (100mW) laser lines are controlled by AOTF.  Collect all images with a Hamamatsu ORCA-ER cooled CCD camera (6.45 µm2 photodiode) with MetaMorph image acquisition software.
?. [Immunofluorescence of autophagic cargo receptors and p-TBK1 at LAMP1 lysosomes during lysophagy]
Display Z series as maximum z-projections and save using Fiji software.
?. [Immunofluorescence of autophagic cargo receptors and p-TBK1 at LAMP1 lysosomes during lysophagy]
Perform the Mander’s Overlap Correlation (MOC) in lysosomes in CellProfiler (see attached CellProfiler pipeline file).
Each field of view for every unique condition is thresholded in the same way. The “identify primary objects” tool is used to find puncta for both the lysosome channel and for the respective receptor or p-TBK1 stain. The “measure colocalization” module is used to compare the fluorescence intensities within the areas defined by the threshold. The MOC with Costes was reported for each field of view..
?. [Immunofluorescence of autophagic cargo receptors and p-TBK1 at LAMP1 lysosomes during lysophagy]
Each channel z series are brightness and adjust contrast equally and then convert to rgb for publication using FIJI software.