Monitoring and assessment of lysosomal membrane damage in cultured cells using the high-content imager

Summary

Autophagy is an intracellular self-degradation process in which part of the cytoplasm, aggregates, or damaged organelles are degraded in lysosomes. Lysophagy is a specific form of selective autophagy responsible for clearing damaged lysosomes. Here, we present a protocol for inducing lysosomal damage in cultured cells and assessing lysosomal damage using a high-content imager and software program. We describe steps for induction of lysosomal damage, image acquisition with spinning disk confocal microscopy, and image analysis using Pathfinder. We then detail data analysis of the clearance of damaged lysosomes.

For complete details on the use and execution of this protocol, please refer to Teranishi et al. (2022).¹

Graphical abstract

Highlights

• •Applicable to various cell lines to investigate lysosomal membrane damage response
• •Detailed steps for inducing lysosomal membrane damages
• •Detection of Galectin-3 signals using the high-content imager
• •Steps to analyze Galectin-3 puncta in images

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Autophagy is an intracellular self-degradation process in which part of the cytoplasm, aggregates, or damaged organelles are degraded in lysosomes. Lysophagy is a specific form of selective autophagy responsible for clearing damaged lysosomes. Here, we present a protocol for inducing lysosomal damage in cultured cells and assessing lysosomal damage using a high-content imager and software program. We describe steps for induction of lysosomal damage, image acquisition with spinning disk confocal microscopy, and image analysis using Pathfinder. We then detail data analysis of the clearance of damaged lysosomes.

Before you begin

The protocol below describes the specific steps for using HeLa cells. For the use of other cell lines, we recommend optimizing experimental conditions. This protocol contains retrovirus production if in need of stable cell lines. For producing retrovirus, ensure to meet the biosafety level of your laboratory before beginning.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Rat monoclonal anti-Galectin-3 (M3/38) (1:1,000 dilution)	Santa Cruz Biotechnology	sc-23938
Mouse monoclonal anti-LAMP1 (H4A3) (1:500 dilution)	Santa Cruz Biotechnology	sc-2001
Mouse monoclonal anti-LAMP2 (H4B4) (1:500 dilution)	Santa Cruz Biotechnology	sc-18822
Alexa Fluor 488 donkey anti-rat IgG	Abcam	ab150153
Chemicals, peptides, and recombinant proteins
Dulbecco’s modified Eagle’s medium (DMEM) -high glucose	Sigma-Aldrich	D6429
Fetal bovine serum (FBS)	Thermo Fisher Scientific	10270-106
Penicillin-Streptomycin	Sigma-Aldrich	P4333
L-Glutamine solution	Sigma-Aldrich	G7513
Trypsin/EDTA	Sigma-Aldrich	T4174
Cellmatrix type I-C	Nitta Gelatin	637-00773
4% paraformaldehyde phosphate buffer solution (4% PFA)	Nakarai Tesque	09154-85
DAPI solution	Nakarai Tesque	19178-91
PEI MAX - Transfection-grade linear polyethylenimine hydrochloride (MW 40,000)	PSI	24765-100
Opti-MEM reduced serum medium	Thermo Fisher Scientific	31985070
Mounting medium with DAPI	VECTASHIELD	H-1200
Leu-Leu methyl ester hydrobromide (LLOMe)	Sigma-Aldrich	L7393
Polybrene	Sigma	H9268
Puromycin	InvivoGen	Ant-pr-1
Experimental models: Cell lines
HeLa Kyoto	Cellosaurus	RRID: CVCL_1922
PlatE	Cell Biolabs, Inc	RV-101 (ref.2)
Software and algorithms
CellPathfinder High-content analysis software	YOKOGAWA	N/A
Excel version: 16.65	Microsoft	N/A
Prism9	GraphPad	https://www.graphpad.com/scientific-software/prism/
Other
6-well plate	Thermo Fisher Scientific	140675
96-well plate (PhenoPlate-96)	PerkinElmer	6055302
6-cm dish	Thermo Fisher Scientific	150462
Millex-HV 0.45μm PVDF 33 mm Gamma Sterilized	Millipore	SLHVR33RB
Micro slide glass	Matsunami	FF-001
Micro cover glass, 12 mm in diameter, Thickness: No.1s (0.16–0.19 mm)	Matsunami	C012001
Filter paper, 3 MM CHR 46 × 57 cm	Whatman	WHA3030917
pMRX-ires-puro-EGFP-Galectin-3 ∗ This plasmid was originally constructed from pEGFP-hGal3.	N/A Addgene	(ref.3) #73080
CellVoyager CQ1 benchtop high-content analysis system (Spinning disk confocal microscope)	YOKOGAWA	Yokogawa cq1

Alternatives: This protocol introduces high-content image analysis using CellVoyager CQ1 and CellPathfinder; using other fluorescent microscopes to assess Gal3-positive signals is possible.

Materials and equipment

Solutions for cell culture and inducing lysosomal damage

Culture medium

Reagent	Final concentration	Amount
Dulbecco’s modified Eagle’s medium (DMEM) -high glucose	N/A	500 mL
Fetal Bovine Serum (FBS)	10%	50 mL
Penicillin-Streptomycin	1%	5 mL
L-glutamine	1%	5 mL
Total	N/A	560 mL

Store at 4°C for up to a month.

10× Phosphate Buffered Saline (PBS)

Reagent	Final concentration	Amount
NaCl	1.37 M	400 g
KCl	27 mM	10 g
Na2HPO4	100 mM	72 g
KH2PO4	18 mM	12 g
ddH2O	N/A	Up to 5 L
Total	N/A	5 L

Adjust pH to 7.4 using HCl or NaOH. Store at 20°C–25°C for up to 3 months.

Note: All PBS used in this protocol are diluted to 1× solution with ddH₂O and autoclaved.

Note: 1× PBS is used at 20°C–25°C unless otherwise stated.

LLOMe solution

Reagent	Final concentration	Amount
LLOMe	1 M	500 mg
DMSO	N/A	1.47 mL
Total	N/A	1.47 mL

Add 1.47 mL of DMSO to the bottle of LLOMe and dissolve completely. Aliquots of LLOMe solution are stored at −20°C or −80°C for up to a year. These aliquots can be re-frozen every time after you use.

Polybrene stock solution

Reagent	Final concentration	Amount
Polybrene	4 mg/mL	400 mg
ddH2O	N/A	100 mL
Total	N/A	100 mL

Filter the solution with 0.45 μm PVDF filter. Aliquots 1 mL filtered solution to tubes and stored at −20°C for up to a year.

Solutions for immunostaining

Blocking buffer

Reagent	Final concentration	Amount
Gelatin	0.2% (w/v)	1 mg
1× PBS	N/A	500 mL
Total	N/A	500 mL

Autoclave and store at 4°C for up to a year.

Digitonin solution

Reagent	Final concentration	Amount
Digitonin	50 mg/mL	50 mg
DMSO	N/A	1 mL
Total	N/A	1 mL

Store at −20°C for up to a year.

Note: Dilute at 1:1000 with 1× PBS before use.

Step-by-step method details

Lysosomes are artificially damaged by treating with L-Leucyl-L-Leucine methyl ester (LLOMe), which becomes membranolytic when LLOMe is cleaved by cathepsin D.⁴ Galectins recognize such lysosome membrane damage, mostly Galectin-3 (Gal3), which binds to N-glycans of proteins by translocating from the cytoplasm to the lysosomal lumen. Thus, we can monitor lysosomal membrane damage, repair, or clearance by observing Gal3 puncta after the washout of LLOMe. Here, we describe two methods to monitor Gal3 puncta using immunostained cells and cell lines stably expressing GFP-Gal3. Our recent studies also show the method and example results.^1,5

Induction of lysosomal damage and detection of the endogenous Galectin-3

Timing: 2 days

• 1.
  Cell culture.

  • a.
    Place sterile coverslips into a 6-well plate (5 coverslips/well).
  • b.
    Coat the coverslips with Cellmatrix Type I-C diluted 10 times with sterile water and incubate the plate for at least 30 min at 20°C–25°C.
  • c.
    Wash the coverslips with PBS twice.
  • d.
    Plate the cells onto the coverslips (1.0–2.0×10⁵ cells/well). The total culture medium should be around 2 mL.

    CRITICAL: Optimize the cell number for your experimental setup. Cells should be at 70%–80% confluency the next day. It is difficult to recognize the cells with CellPathfinder (See step 8) if the cells are almost confluent.

  • e.
    Incubate the cells with 5% CO₂ for 18–24 h at 37°C.
• 2.
  Induction of lysosomal membrane damage.

  • a.
    Add 2 mL of LLOMe containing culture medium at 1 mM final concentration.

    Note: Optimize the LLOMe concentration for your cell lines.

  • b.
    Incubate the cells for 1 h at 37°C.
  • c.
    Remove the LLOMe solution, and then wash the cells gently with 2 mL of PBS twice.
  • d.
    Add 2 mL of fresh culture medium.
  • e.
    Incubate the cells for 1 or 10 h at 37°C.

    Note: Optimize the incubation time for your experimental setup.

  • f.
    Wash the cells with 2 mL of PBS twice.
  • g.
    For fixation, remove PBS, add 1 mL of 4% PFA and incubate the cells for 20 min at 20°C–25°C under aluminum foil.

    Note: PFA solution should be equilibrated to 20°C–25°C before use.

  • h.
    Wash the cells with 2 mL of PBS twice.

    Pause point: After the PFA fixation, the samples can be stored in PBS at 4°C for a week.

• 3.
  Immunostaining for endogenous Galectin-3.

  • a.
    Permeabilize the cells with 50 μg/mL digitonin-PBS solution for 10 min at 20°C–25°C.
  • b.
    Wash the samples with PBS twice.
  • c.
    For blocking, incubate with 0.2% gelatin-PBS for 30 min at 20°C–25°C.
  • d.
    Wash the samples with PBS twice.
  • e.
    Incubate the cells with 40 μL/coverslip of anti-Galectin-3 primary antibody solution diluted at 1: 1,000 with 0.2% gelatin-PBS solution for 1 h at 20°C–25°C.

    Note: You should include anti-LAMP1 or LAMP2 antibody to stain lysosomes as a control. See troubleshooting, problem 5.

    Pause point: The samples can be incubated at 4°C for 14–18 h.

  • f.
    Wash the samples with PBS for 5 min three times.
  • g.
    Incubate the cells with 40 μL/coverslip of the secondary antibody solution diluted at 1: 500 with 0.2% gelatin-PBS solution for 40–60 min at 20°C–25°C.

    CRITICAL: The samples should be under aluminum foil to prevent bleaching.

  • h.
    Wash the samples with PBS for 5 min three times.
  • i.
    Drop 5–10 μL/coverslip of the mounting medium with DAPI (e.g., VECTASHIELD H-1200) onto glass slides and mount the coverslips at cell surface-down.
  • j.
    Remove the excess mounting medium with filter papers and seal the coverslips with nail polish.

    Pause point: After mounting, the samples can be stored in a black box at 4°C for a month.

Induction of lysosomal damage and detection of GFP-Gal3

Timing: 2–10 days

• 4.
  Cell culture (in case you need to establish cell lines stably expressing GFP-Gal3).

  Note: Go to step 5 if you have cells stably expressing GFP-Gal3.

  Note: Check the biosafety level of your laboratory to produce infectious retrovirus.

  Note: Transient highly overexpression may induce protein aggregates. These GFP-positive aggregates cannot be distinguished with GFP-Gal3-positive damaged lysosomes after LLOMe treatment. Thus, we recommend using stably mild-expressing cells in which GFP-Gal3 should be distributed to cytoplasm without puncta. However, transient expression can also be used if GFP-Gal3 is distributed to cytoplasm in plasmid-transfected cells.

  • a.
    Cell plating.

    • i.
      Plate PlatE cells at 2.0 × 10⁶ cell/dish in a 6 cm dish with culture medium described in Materials. The total culture medium is 2 mL.
    • ii.
      Incubate the cells with 5% CO₂ for 18–24 h at 37°C.
  • b.
    Transfection for retrovirus production.

    • i.
      Tube 1: Mix 3 μg of pMRX_IRES-puro_GFP-Gal3 and 3 μg of pVSVG in 250 μL of OPTI-MEM.
    • ii.
      Tube 2: Add 16 μL of PEI to 250 μL of OPTI-MEM and mix well.
    • iii.
      Mix tube-1 and tube-2 and incubate for 15 min at 20°C–25°C.
    • iv.
      Drop 500 μL of the mixture into 2.5 mL fresh culture medium.
    • v.
      Gently shake the dish to mix and incubate the cells with 5% CO₂ for 24 h at 37°C.
    • vi.
      Change to fresh culture medium and incubate the cells with 5% CO₂ for 24 h at 37°C.

      Note: The culture medium contains retrovirus. Cells and the medium must be handled in a biosafety cabinet.

  • c.
    Retrovirus infection.

    • i.
      One day before infection, plate HeLa cells at 1.0 × 10⁵ cells/well in a 6-well plate.

      Note: If cell confluency on infection day is 100%, transduction efficiency becomes quite lower. The ideal confluency on the infection day is around 50%–80%.

    • ii.
      At 48 h post-transfection, the culture medium is filtered with a sterilized 0.45 μm PVDF filter to remove cell debris.

      Pause point: The virus-containing medium can be stored at −80°C for a month, although the virus titer may decrease 10–100-fold.

    • iii.
      Mix 200–500 μL of virus-containing medium with 2 μL of 4 mg/mL polybrene.
    • iv.
      Drop the mixture into 2 mL of fresh culture medium in the 6-well plate.
    • v.
      Incubate the cells with 5% CO₂ at 37°C for 14–18 h.
  • d.
    Cell selection.

    • i.
      Replace to medium containing 3 μg/mL of puromycin.
    • ii.
      Incubate and select the cells.

      Note: Cells without an infection should die after 3–5 days in a culture medium containing puromycin.

      Note: You do not need to pick up single clone if you have stably mild-expressing cells in which GFP-Gal3 should be distributed to cytoplasm without puncta.

• 5.
  Cell culture.

  • a.
    Add 100 μL of culture medium into each well of 96-well glass bottom plate.

    Note: More than 3 wells for one sample condition are needed to analyze statistically.

  • b.
    Plate 100 μL of cell suspension into a 96 well-plate at 4,000 cells/100 μL and leave the plate for 15 min at 20°C–25°C until cells sink to the bottom.
  • c.
    Incubate the cells with 5% CO₂ for 18–24 h.

    CRITICAL: After this plating step, cell distribution can be problematic. Take a close look at the cell distribution before you start the next experiment. See troubleshooting, problem 2.

• 6.
  Induction of lysosomal membrane damage.

  • a.
    Add 100 μL of LLOMe containing culture medium at 1 mM final concentration.

    Note: Optimize the LLOMe concentration for your cell lines.

  • b.
    Incubate for 1 h at 37°C.
  • c.
    Remove the medium and wash the cells with 200 μL of PBS twice.

    CRITICAL: DO NOT pipette vigorously. Add or aspirate the liquid gently not to detach the cells.

  • d.
    Add 200 μL of fresh medium to the cells.
  • e.
    Incubate the cells for 1 or 10 h at 37°C.

    Note: Optimize the incubation time for your experimental setup.

  • f.
    Wash the cells with 200 μL of PBS twice.
  • g.
    For fixation, remove PBS, add 100 μL of 4% PFA and incubate the cells for 20 min at 20°C–25°C under aluminum foil.

    Note: PFA solution should be equilibrated to 20°C–25°C before use.

  • h.
    Remove 4% PFA and wash the cells with 200 μL of PBS twice.

    Pause point: After PFA fixation, the samples can be stored in PBS at 4°C for a week.

    Note: The plate should be covered with aluminum foil to avoid bleaching.

  • i.
    Remove PBS, add 100 μL of DAPI solution diluted at 1: 2,000 with PBS, and incubate the samples for 30 min at 20°C–25°C.

    CRITICAL: The plate should be covered with aluminum foil to avoid bleaching.

  • j.
    Remove the DAPI solution and wash the cells with 200 μL of PBS twice.

    Pause point: The samples can be stored in PBS at 4°C within a week.

    CRITICAL: The plate should be covered with aluminum foil to avoid bleaching.

Acquisition of microscope images with CellVoyager CQ1

Timing: 1–2 h

CellVoyager CQ1 enables us to acquire massive datasets semi-automatically at high speed. Even when we use a 96 well-plate, we can take images from multi spots in a well. This microscopy leads to unbiased results.

Note: Other fluorescent microscopes are also available for taking cell images manually.

Note: Refer to an instruction book from YOKOGAWA as well.

• 7.
  Image acquisition with CellVoyager CQ1.

  • a.
    Set the glass slides from step 3j or the 96 well-plate from step 6j to the sample holder.
  • b.
    Open the software “CQ1 Measurement”.
  • c.
    Select a saving folder using the “Save Setting” tab (Figure 1A).
  • d.
    Select the wavelengths (Figure 1B).
  • e.
    Adjust focus using the “Search” button (Figure 1C).

    Note: Set range to ∼1.0.

    Note: After “Search” completes, click the “<<” button to find the in-focus position. If the image is still out of focus, alter “Center [μm]” little by little to adjust focus.

    CRITICAL: If you mount coverslips on glass slides, adjust the focus manually for each coverslip.

  • f.
    Set photographing areas (Figure 1D).
  • g.
    Start capturing images by pushing the “Rec” button (Figure 1E).

Figure 1.

Setting screens of CellVoyager CQ1, related to step 7

(A–E) CQ1 setting screens for (A) saving folder, (B) wavelength, (C) focus, and (D) shooting areas. The “Rec” button in (E) starts photographing.

Image analysis with CellPathfinder

Timing: 1–2 h

• 8.
  Image analysis using CellPathfinder.

  Note: In case you analyze and evaluate images obtained with other types of microscopes, refer to the section of step 9.

  • a.
    Open “CellPathfinder” software.
  • b.
    Click the “Data” tab and load the image datasets acquired in step 7 (Figure 2A).
  • c.
    Set the “Channels” you used in step 7 (Figures 2B and 2C).
  • d.
    Set “Object” to be recognized (Figures 2B and 2D).

    Note: The same channel can recognize the cytoplasm as Gal3.

  • e.
    Set “Algorithm” to recognize the nuclei, the cytoplasm, and the Gal3-positive puncta by following procedures (See Figure 2B and Table 1).

    • i.
      Set parameters to recognize the nuclei from DAPI signals (See Table 2).
    • ii.
      Set parameters to recognize the cytoplasm (See Figure 2E and Table 3).
    • iii.
      Identify the Gal3-positive puncta (See Figure 2F and Table 4).

      Note: You can check the detection range of each step by clicking the button.

  • f.
    Set “Link” (Figures 2B and 2G, Table 5).

    Note: The nucleus defines the cytoplasm because one cell has one nucleus. Gal3 forms large numbers of dots in the cytoplasm; thus, “Gal3” should be linked to “Cytoplasm” using “Include” and “1 to Many” (See Table 5).

  • g.
    Determine the Analysis Range (Figure 2H).
  • h.
    Click “Feature” and select the datasets such as Size and Count (Figures 2B and 2I).
  • i.
    Start the analysis with the “Start” button (Figure 2B).
  • j.
    Export data as “CSV” (Figure 2J).

    Note: After the analysis completes, the recognized objects are viewed on the screen (Figure 2K).

Figure 2.

Setting screens of CellPathfinder, related to step 8

(A, C, D, G, H, and I) CellPathfinder setting screens for (A) analysis datasets, (C) analysis channels, (D) assemble objects, (G) links, (H) analysis range, and (I) features.

(B and J) Show the location of the respective setting buttons.

(E and F) The areas encircled by the lines indicate the cytoplasm (E) or Gal3 dots (F).

(K) After the completion of the analysis, the recognized objects are confirmed in the “image” tab.

Table 1.

Description of parameters in CellPathfinder

Parameter	Explanation
Gaussian	Smooth the image to reduce the image noise.
Threshold	Binarize the image and extract the regions with greater intensity than a defined threshold.
DynamicThreshold	Mask Size: Determine the size of the area to be extracted. Recommended value: Twice as large as the object’s diameter to be recognized.
	Detect Factor: Determine the detection sensitivity. It is recommended to set the value around 1.0.
	Min. Gray Offset: Determine the threshold. This value must be larger than the background intensity.
OrImage	Extract the union of the two regions.
ClosingCircle	Fill up a hole and make the object a round shape.
OpeningCircle	Eliminate protrusion and small noises to make the object a round shape.
FindMaximamDistance	Minimum Point Distance [μm]: Determine the minimum distance between the extreme points. Recommend setting this value as large as the average diameter of the objects. Note: The extreme point is defined as a site that is farthest from the background.
	Remove Size [μm]: Determine the diameter of the area to be excluded from the recognition range.
DilationCircle	Expand the region roundly.
Labeling	Label the adjacent pixels to separate them into individual regions.
SizeFilter	Determine the area or volume to be recognized, and exclude the objects of abnormal size.
ExpandRegion3D	Using two labeled regions, expand the former region within the latter region. If the two regions do not overlap, the latter region is excluded.
CompactnessFilter(2D)	Exclude the objects which have an abnormal shape. Note: Compactness approaches 1 when the object is close to a perfect circle. If too many abnormal shapes are recognized, this value should be lowered.
ExcludeEdge	Exclude the objects on the edge of the image.
ErodeRegion	Shrink the labeled range. Note: this process allows the extraction of the dots at the interface between the nucleus and the cytoplasm.
SubRegion	Extract the former region minus the latter region.

Brief explanation of parameters necessary for the image analysis with CellPathfinder. These parameters are used in step 8e.

Table 2.

Setup to recognize nuclei in CellPathfinder

Parameter				Note
ImageFIlter	[1] Gaussian	IN Previous Output	Mask Size (μm)	Set to 1.0 μm for HeLa cells.
Binarization	[2] DynamicThreshold	IN Previous Output	Mask Size (μm)	–
			Detect Factor	–
			Min. Gray Offset [gray level]	–
BinarizeTransform	[3] ClosingCircle	IN Previous Output	Diameter (μm)	Adjust the value depending on the staining intensity.
	[4] OpeningCircle	IN Previous Output	Diameter (μm)	–
	[5] Find MaximumDistance	IN Previous Output	Minimum Point Distance (μm)	–
			Remove Size (μm)	–
	[6] DilationCircle	IN Previous Output	Diameter (μm)	Set to 5.0.
Labeling	[7] Labeling	IN Previous Output	–	–
	[8] Labeling	IN Nucleus [4] OpeningCircle	–	–
LabelTransform	[9] ExpandRegion3D	IN Nucleus [7] Labeling, Nucleus [8] Labeling	–	–
	[10] CompactnessFilter(2D)	IN Previous Output	Range	Recognize the nuclei with abnormal shapes.
	[11] SizeFilter	IN Previous Output	Range	Adjust the value depending on the cell type or the treatment.
	[12] Subregion	IN Nucleus [9] ExpandRegion-3D, Nucleus [10] Compactness-Filter(2D)	–	Exclude the nuclei with abnormal shapes.
	[13] ErodeRegion	IN Previous Output	Length (μm)	Recognize the dots at the interface between the nucleus and the cytoplasm.

This table shows how to set algorithm for the recognition of nuclei. See step 8e i.

Table 3.

Setup to recognize cytoplasm in CellPathfinder

Parameter				Note
ImageFilter	[1] Gaussian	IN Previous Output	Mask Size (μm)	Set to 1.0 μm for HeLa cells.
Binarization	[2] Threshold	IN Previous Output	Threshold [gray level]	Adjust the value depending on the luminance of the background and the cytoplasm.
BinarizationTransform	[3] ClosingCircle	IN Previous Output	Diameter (μm)	–
	[4] OpeningCircle	IN Previous Output	Diameter (μm)	–
Labeling	[5] Labeling	IN Previous Output	–	–
LabelTransform	[6] ExpandRegion3D	IN Nucleus [9] ExpandRegion3D, cytoplasm [5] Labeling	–	–
	[7] ExcludeEdge	IN Previous Output	Range	Exclude the cells on the edge of the image.
	[8] ErodeRegion	IN Previous Output	Range	Shrink the recognized area to separate the individual cells, which are closely attached.
	[9] ExpandRegion3D	IN Nucleus [9] ExpandRegion-3D, Nucleus [10] Compactness-Filter(2D)	–	–
	[10] SubRegion	IN Cytoplasm [8] ErodeRegion, Cytoplasm [9] ExpandRegion3D	–	–
	[11] SubRegion	IN Previous Output, Nucleus Result	–	Exclude the nucleus region from the entire cell body.
	[12] SizeFilter	IN Previous Output	Range	–

This table shows how to set algorithm for the recognition of cytoplasm. See step 8e ii.

Table 4.

Setup to recognize Gal3-positive dots in CellPathfinder

Parameter				Note
ImageFilter	[1] Gaussian	IN Previous Output	Mask Size (μm)	–
Binarization	[2] Threshold	IN Previous Output	Threshold [gray level]	–
	[3] DynamicThreshold	IN Gal3 [1] Gaussian	Mask Size (μm)	Set to a similar number with a diameter of one dot.
			Detect Factor	Set to less than 1.0. The noise increases when set to 1.0 or above.
			Min. Gray Offset [gray level]	–
BinarizationTransform	[4] OrImage	IN Gal3 [3] Dynamic-Threshold, Gal3 [4] Threshold	–	–
Labeling	[5] Labeling	IN Previous Output	–	–
LabelTransform	[6] SizeFilter	IN Previous Output	Range	–
	[7] SubRegion	IN Previous Output, Nucleus Result	–	Recognize the dots located in the cytoplasm.

This table shows how to set algorithm for the recognition of Gal3-positive dots. See step 8e iii.

Table 5.

Description of symbols in CellPathfinder

Method	Explanation
Neighbor	Associate a child object adjacent to a parent object.
Include	Associate a child object included in a parent object.
1 to 1	Link a single child object to a single parent object. Note: Since one cell has a nucleus, “Cytoplasm” must be associated with “Nucleus” using “1 to 1" (step 8f and Figure 2G).
1 to Many	Link several objects to a single parent object. Note: Since a lot of Gal3 dots exist in a cell, “Gal3” must be associated with “Cytoplasm” using “1 to Many” (step 8f and Figure 2G).

Explanations for the symbols in the setting “Link”. See step 8f.

Data analysis

Timing: 1 h

• 9.Data analysis and assessment of the clearance of damaged lysosomes (Figure 3).
  Based on a dataset from step 8, this step describes how to analyze and quantify the remaining Gal3-positive puncta 10 h after LLOMe treatment.

  CRITICAL: Gal3 puncta occasionally accumulate at perinuclear region and make a cluster. The numbers of Gal3 puncta may be fewer than the actual numbers when counted by CellPathfinder or other software. Thus, we recommend analyzing and showing as (GFP-)Gal3-positive area per cell in a final graph.

  Note: At least three independent data sets are needed for statistical analysis.

  • a.
    Open the data file acquired in step 8.
  • b.
    Copy and paste “WellID”, “[Cell1] Count”, “[Cell1] (Cytoplasm) Area (AVG)”, and “[Cell1] (Gal3 (Cytoplasm)) Area TOTAL (AVG)” to the Excel sheet (Figure 3, column A-B and E-F).
  • c.
    To show the area of Gal3 per cytoplasm, divide [Cell1] (Gal3 (Cytoplasm)) Area TOTAL (AVG) by [Cell1] (Cytoplasm) Area (AVG) (Figure 3, column G).
  • d.
    Calculate the average of values from each well of 1 h-samples (Figure 3, cell J8 and K8).
  • e.
    Divide the values from 10 h-samples by the average score form 1 h-samples (Figure 3, cell J2-6 and K2-6).
  • f.
    Make a graph showing the Gal3-positive area per cell.
  • g.
    Test significant differences with GraphPad Prism.

Figure 3.

Quantification of lysosomal damage, related to step 9

The Excel sheet shows how to quantify the lysosomal damage from the analysis datasets acquired by CellPathfinder.

Expected outcomes

In a steady state, Gal3 is diffused in the cytosol and does not form puncta (Figure 4A, left). LLOMe-induced lysosomal damage enables Gal3 to access the luminal side of lysosomes. Thus, Gal3 puncta are observed at 1 h after LLOMe treatment (Figure 4A, middle) and decreased after 10 h incubation (Figure 4, upper right). In the autophagy-deficient cells, such as Atg9A knockout cells, Gal3 puncta remain compared to the controls (and Figure 4A, lower right, and Figure 4B).

Figure 4.

Monitor Galetcin-3 as an indicator of damaged lysosomes

HeLa cells stably expressing GFP-Gal3 were infected with lentivirus containing sgRNAs to construct indicated knockout cells. 7 days later, the cells were treated with 1 mM LLOMe for 1 h. After LLOMe washout, the cells were incubated for 1 or 10 h.

(A) Representative images are shown. Scale bars, 20 µm.

(B) The graph represents mean ± SD from 5 wells. An unpaired t test was used to test the significant difference.

Limitations

The treatment with LLOMe is optimized for HeLa cells in this protocol. LLOMe is toxic for cells and may induce cell death in other cell lines. In our experience, the LLOMe treatment at a higher concentration of more than 1 mM for 1 h affected cell viability. Please optimize the final concentration or treatment time of LLOMe for the cell line used.

Whereas LLOMe treatment injures lysosomal membranes, polystyrene beads coated with Effectene, transfection reagent, and silica crystals injure both endosomal membranes and lysosomes. Gal3 is also recruited to damaged endosomes and detected as Gal3-positive puncta. When using other reagents to induce endosomal/lysosomal membrane damage, careful interpretation of results is required.

After washout of LLOMe, Gal3 puncta is decreased and returns to the cytoplasmic pattern of the pre-treatment state. This result indicates that lysophagy removes damaged lysosomes. However, in the use of autophagy-deficient cells, the area of Gal3 puncta reduces over time, suggesting that there are other repair types of machinery exist, like ESCRT-III.^6,7 Additional experiments are needed to distinguish these responsible molecular mechanisms.

LLOMe treatments with different conditions, such as concentration or treatment time, may induce different degrees or sizes of lysosomal membrane injury.^6,7,8 Based on these membrane injuries, distinct molecules may be recruited to repair or remove damaged lysosomes.

Troubleshooting

Problem 1

The cell number in a well is inappropriate (steps 1 or 5).

Potential solution

The cell number per well highly depends on cell lines, culture medium, incubation period, and coating reagents. Optimize the cell number for each experimental setup. If the cells are too few or confluent when imaging; analysis cannot be carried out accurately, thus affecting the final results.

Problem 2

Uneven plating of cells (steps 1 or 5).

Potential solution

First, prepare cell suspension and pour the solution slowly and carefully into a well. We do not recommend mixing a high concentration of cell suspension with a culture medium in a small well; cells that are one-sided or centered in the well could affect final results.

Problem 3

Cells die after LLOMe treatment (steps 2e or 6e).

Potential solution

Optimize the concentration of LLOMe for each cell line. The lower concentration or shortened time of the LLOMe treatment less affects cell viability.

Problem 4

Cells detach from the bottom of the culture plate (steps 2e or 6e).

Potential solution

First, optimize the concentration of LLOMe for each cell line. Alternatively, aspirate or add solution slowly and carefully using well wall when you change the medium or wash cells. Although coating the coverslips or the 96 well-plate with Cellmatrix Type I-C or other reagents is not mandatory in some cell lines, it helps cells adhere tightly to the bottom of the plate.

Problem 5

Gal3 staining does not work (step 3).

Potential solution

If you have technical concerns in immunostaining against Gal3, we recommend staining LAMP1 or LAMP2 as a control. Primary antibodies for them can be diluted to 1:500.

If you need to confirm lysosomal membrane damage after LLOMe treatment, you can observe co-localization between Gal3 and LAMP1 or LAMP2. Basically, you can use same protocols for image acquisition with CQ1 and image analysis with CellPathfinder. For multi-color imaging and analysis, you need to add another wavelength in CQ1 and channel in CellPathfinder.

Problem 6

CellPathfinder does not correctly recognize nuclei, cytoplasm area, or Gal3 puncta (step 8e).

Potential solution

In capturing images with CQ1, increase the laser power or the exposure time to make the objects more luminous. Also, optimize the parameters in CellPathfinder, particularly in Threshold/DynamicThreshold, ClosingCircle, OpeningCircle, and SizeFilter (See Table 1, Table 2, Table 3, Table 4).

Problem 7

Gal3 puncta remain 10 h after LLOMe treatment even in control samples (step 9).

Potential solution

Optimization of the experimental setups, such as the concentration of LLOMe, the treatment time, and time points of fixation after washout, is required (e.g., until 10, 24 h). Alternatively, treatment with si/sh/sgRNA against the control target or transfection reagents might affect the assay. It would be better to exclude the off-target possibility by comparing it with proper control samples.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact Maho Hamasaki (hamasaki@fbs.osaka-u.ac.jp).

Materials availability

This study did not generate new unique reagents.

Data and code availability

This study did not generate and analyze any original datasets/codes.