Protocol for detecting lysosome quantity and membrane permeability in acute myeloid leukemia cell lines

Summary

Lysosomal function and activity are essential to support cellular adaptation to multiple stresses. For example, certain drugs can induce increased lysosomal membrane permeability to exert their anti-cancer effects. Here, we present a protocol to evaluate the lysosome alterations induced by drug treatment. We first describe the steps for inducing lysosomal alterations in cultured cells. We then show how to quantify the number of lysosomes, assess the integrity of lysosomal membranes, and determine lysosomal membrane permeabilization by using galectin puncta assay.

For complete details on the use and execution of this protocol, please refer to Jiang et al.¹

Graphical abstract

Highlights

• •Protocol to assess lysosome alterations in AML cells
• •Procedures for cell seeding and lysosomal fluorescent staining
• •Construction of LGALS3-EGFP cell line
• •Detection of lysosomal membrane permeabilization via galectin assay

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

Lysosomal function and activity are essential to support cellular adaptation to multiple stresses. For example, certain drugs can induce increased lysosomal membrane permeability to exert their anti-cancer effects. Here, we present a protocol to evaluate the lysosome alterations induced by drug treatment. We first describe the steps for inducing lysosomal alterations in cultured cells. We then show how to quantify the number of lysosomes, assess the integrity of lysosomal membranes, and determine lysosomal membrane permeabilization by using galectin puncta assay.

Before you begin

The ability to detect lysosome alterations can be useful for exploring mechanisms of lysosomal homeostasis (repair, lysophagy, and biogenesis) in steady-state, lysosomal destabilization under pathological conditions, and lysosome-dependent cell death after therapies. In this protocol, we aim to evaluate the lysosome alterations including lysosome number and lysosomal membrane permeability.

Despite the diminutive dimensions and suspension properties characteristic of hematological malignant cells, which pose considerable obstacles to the direct microscopic observation of their morphological features and specific subcellular structures, such as lysosomes, we have devised a facile and effective assay for evaluating lysosomal activity and membrane integrity in suspension cell populations. We describe the specific steps for using acute myeloid leukemia (AML) cell line MV4-11, and also use this protocol in other suspension cell lines (e.g., MOLM13, THP-1), demonstrating its utility in the investigation of lysosome alterations within these models.

This protocol can be adapted to investigate the lysosomal alterations induced by pharmacological inhibition in other suspension cells, serving as the easily accessible method before further molecular mechanism studies involving the regulation of lysosome homeostasis.

Note: This protocol contains lentivirus production for establishing stable cell lines. Hence, ensure to work in a Biosafety Level 2 (BSL-2) laboratory with appropriate personal protective equipment (PPE) before beginning.

Cell culture and drug treatment

Timing: 1–2 weeks

• 1.
  Thawing cells.

  • a.
    Quickly thaw the cryopreserved cell vial in a 37°C water bath.
  • b.
    Gently agitate the vial to evenly thaw the cells.
  • c.
    Once thawed, promptly transfer the cell suspension to a sterile centrifuge tube containing 9 mL pre-warmed culture medium.
• 2.
  Centrifugation and resuspension.

  • a.
    Centrifuge the tube at 300 ×g for 5 min at 32°C to pellet the cells.
  • b.
    Carefully aspirate and discard the supernatant.
  • c.
    Resuspend the cells in an appropriate volume of fresh, pre-warmed culture medium to achieve a final cell density of 10⁶ cells/mL.
• 3.
  Culturing.

  • a.
    Transfer the cell suspension to a sterile culture flask or dish and place the flask in a 37°C incubator with 5% CO₂.
  • b.
    Assess the condition of the cells by morphology and background or Trypan Blue exclusion after 24 h.

    Note: Healthy suspension cells should maintain their characteristic size and round shape with smooth edges. Cell clumping, swelling, shrinking, or aggregation can be signs of poor health. Normally, the culture medium is clear and transparent. Background with cell debris or fragments under a microscope also indicates suboptimal conditions. Figure 1 depicts the morphology of healthy MV4-11 cells observed under a light microscope.

  • c.
    Transfer the cell suspension to a sterile centrifuge tube and centrifuge at 300 ×g for 5 min.
  • d.
    Discard the supernatant and resuspend the cell pellet in a fresh medium to achieve the optimal seeding density.

    Note: Seeding density depends heavily on the cell line. For this protocol, we used MV4-11 and chose a final density of 0.5–1×10⁶ cells/mL. For other fast-growing cells like HL-60 and U937, lower initial densities may be required.

• 4.
  Maintenance.

  • a.
    Return the cells to the incubator and continue with regular monitoring and maintenance.
  • b.
    Change the medium every 2–3 days or as required.

    Note: The frequency of medium change depends greatly on the specific characteristics and requirements of the cell type being cultured. For example, THP-1 cells are known to be quite adaptable and tolerate slightly acidic conditions, medium changes can be extended to 3 days.

  • c.
    Subculture the cells upon reaching 80%–90% confluency.
  • d.
    Conduct experiments when cells are in their exponential (log) growth phase.

Figure 1.

An example of morphologically healthy MV4-11 cells observed under a light microscope

Determining the conditions to induce lysosomal alterations

Timing: 3–4 days

Before the final detection, we recommend determining the optimal condition to induce lysosomal alterations. Several types of compounds/drugs are used in research to study lysosomal function and related cellular processes, and known candidates include chloroquine (or hydroxychloroquine), V-ATPase inhibitor Bafilomycin A1, and lysosomal damage model. In this protocol, we treat AML cells, MV4-11 for example, with hexamethylene amiloride (HA), a lysosomotropic agent, combined with venetoclax (Ven) to induce lysosomal alterations. The synergistic therapeutic strategy involving HA and Ven has been demonstrated to induce the death of leukemia cells by modulating an increase in lysosomal abundance and compromising lysosomal membrane integrity.¹ We recommend choosing the conditions that cause around 50% of cell viability/cell death. This ensures enough live cells to demonstrate potential changes in lysosomes, while allowing the evaluation of the cytotoxic effects induced by lysosomal alterations.

• 5.
  Seed cells.

  • a.
    Plate MV4-11 cells at 1.0×10⁵ cells/well in a 6-well plate.
  • b.
    Incubate the plates in a CO₂ incubator at 37°C with 5% CO₂ to allow cell growth for 24 h.
• 6.Prepare working solutions of HA at a concentration of 10 mM and Ven at a concentration of 0.05 mM in sterile DMSO.
• 7.Add 3 μL HA solutions and 3 μL Ven solution in a 3 mL culture system to achieve the final concentration (10 μM HA and 0.05 μM Ven).
• 8.Pat the plate gently to ensure an even mixing of the drug and the cells.
• 9.Incubate the treated cells for 48 h.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Chemicals, peptides, and recombinant proteins
Hexamethylene amiloride	MedChemExpress	Cat#HY-128067
Venetoclax	MedChemExpress	Cat#HY-15531
Acridine orange hydrochloride	MedChemExpress	Cat#HY-101879
LysoTracker Deep Red	Thermo Fisher Scientific	Cat#L12492
RPMI1640	Gibco	Cat#C11875500BT
DMEM	Gibco	Cat#11965118
Fetal bovine serum	Biological Industries	Cat#04-001-1ACS
Trypan blue	Solarbio	Cat#C0041
DMSO	Solarbio	Cat#D8371
Penicillin-streptomycin	Gibco	Cat#15140122
Lipo 3000	Thermo Fisher Scientific	Cat#L3000001
Polybrene	Biosharp	Cat#BL628A
Puromycin	Beyotime	Cat#ST551
4% paraformaldehyde	Biosharp	Cat#BL539A
CCK-8	Yeasen	Cat#40203ES80
DAPI	Sigma-Aldrich	Cat#D9542
Antifade mounting medium	Biosharp	Cat#BL701A
Critical commercial assays
EndoFree Mini Plasmid Kit II	TIANGEN BIOTECH	Cat#DP118-02
Experimental models: Cell lines
Human: MV4-11 cell line	ATCC	Cat#CRL-9591
Recombinant DNA
Plasmid: EGFP-Homo-LGALS3	IGEbio	N/A
Plasmid: psPAX2	Gifted from Dr. Jinyong Wang, Guangzhou Institutes of Biomedicine and Health	N/A
Plasmid: pMD2.G	Gifted from Dr. Jinyong Wang, Guangzhou Institutes of Biomedicine and Health	N/A
Software and algorithms
FlowJo Software	FlowJo	https://www.flowjo.com/solutions/flowjo/downloads
Zeiss LSM880 Airyscan confocal microscope	Zeiss	https://www.zeiss.com.cn/microscopy/products/microscope-software.html
DxFLEX	Beckman Coulter	https://www.mybeckman.cn/en/flow-cytometry/clinical-flow-cytometers/dxflex
GraphPad Prism 8.0	GraphPad Prism	https://www.graphpad.com/scientific-software/prism
Other
Centrifuge 5418 R	Eppendorf	Cat#5401000099
Centrifuge 5810 R	Eppendorf	Cat#5811000096
Eppendorf S-4-104 rotor	Eppendorf	Cat#5820740000
CO2 incubators	Thermo Fisher Scientific	Cat#311
60 mm culture dish	Corning	Cat#430166
1.5 mL EP tubes	Axygen	Cat#MCT-150-C
15 mL centrifuge tubes	NEST	Cat#601052
50 mL centrifuge tubes	NEST	Cat#602052
Flow cytometry tubes	Biosharp	Cat#BS-PST-5-G-S1
0.45 μm sterile syringe filter	Corning	Cat#431225
Slides coated with poly-L-lysine	Merck Millipore	Cat#P0425

Materials and equipment

Cell culture medium

Reagent	Final concentration	Amount
RPMI-1640/DMEM	89%	445 mL
Fetal Bovine Serum (FBS)	10%	50 mL
Penicillin-Streptomycin	1%	5 mL
Total	N/A	500 mL

Store at 4°C for up to 1 month

Note: The type of basic culture medium should be based on the cell type.

HA stock solution

• •Weighing the Drug: Accurately weigh 1 mg of HA drug powder.
• •Dissolve the weighed HA drug in 320.7 μL of sterile DMSO to create a stock solution (10 mM).
• •Aliquoting: Dispense the resulting solution into sterile EP tubes.

Store at −20°C for 1 year and −80°C for 2 years.

Ven stock solution

• •Weighing the Drug: Accurately weigh 1 mg of Ven drug powder.
• •Dissolve the weighed Ven drug in 23.03 mL of sterile DMSO to create a stock solution (0.05 mM).
• •Aliquoting: Dispense the resulting solution into sterile EP tubes.

Store at −20°C for 1 year and −80°C for 2 years.

Step-by-step method details

Lysotracker staining

Timing: 1 h 30 min

Lysotracker Deep Red is a cell-permeable, red fluorescent dye that stains acidic compartments within a cell, such as lysosomes.² In this step, the number of lysosomes is quantified by lysotracker staining using flow cytometry.

• 1.
  Working solution preparation.

  • a.
    Dilute the Lysotracker Deep Red (1 mM) reagent with PBS to 10 μM.

    Note: The diluted reagents can be stored at −20°C in the dark for more than 1 month.

  • b.
    Add Lysotracker Deep Red (10 μM) reagent to the cell culture medium at a ratio of 1:200 to achieve a final concentration of 50 nM.
  • c.
    Equilibrate the working solution to 37°C before use.

    Note: The concentration of Lysotracker Deep Red in the working solution should be appropriately adjusted. Pre-testing of the extent of potential lysosome quantity alteration is needed. Increase the concentration or dilute the cells if drug treatment increases lysosomal quantity extensively. It is recommended to use as low a concentration of Lysotracker Deep Red as possible. Serial concentrations can be tested and the lowest concentration to yield a peak shift is recommended for subsequent tests.

• 2.
  Harvesting of the cells.

  • a.
    Collect the cultured cells into a 15 mL centrifuge tube.
  • b.
    Centrifuge the 15 mL tube at 300 ×g for 5 min.
  • c.
    Discard the supernatant and resuspend these cells with 5 mL of PBS.
  • d.
    Centrifuge the 15 mL tube at 300 ×g for 5 min.
  • e.
    Discard the supernatant.
• 3.
  Staining with Lysotracker Deep Red and DAPI.

  • a.
    Resuspend these cells with Lysotracker Deep Red working solution.

    Note: The volume of the working solution is determined according to the number of cells. To quantify the number of lysosomes by flow cytometry, we recommend 2×10⁵ cells to be resuspended in 200 μL of the working solution.

  • b.
    Add DAPI at a final concentration of 5 μg/mL.
  • c.
    Incubate the cells for 15 min to 1 h at 37°C in the dark.

    CRITICAL: This reagent can stain other acidic organelles, and it is recommended that the lowest but sufficient concentrations should be used to preferentially stain acidic lysosomes. The lowest concentration pre-determined in step 1b to yield a peak shift is recommended.

  • d.
    Stop the reaction with 1 mL of PBS.
• 4.
  Measurement and analysis.

  • a.
    Collect the stained cells in a flow cytometry tube.
  • b.
    Centrifuge the tube at 300 ×g for 5 min.
  • c.
    Discard the supernatant and resuspend these cells with 400 μL of PBS.
  • d.
    Measured the lysosomal fluorescence by using DxFLEX (Beckman Coulter). Flow cytometry gating strategy to obtain mean fluorescence intensity (MFI) of lysotracker staining is shown in Figure 2A.

    Note: The maximum excitation and emission wavelength of the Lysotracker Deep Red stain was 647/668 nm. We can use the APC channel for detection.

    Note: It is recommended to detect fluorescence immediately after staining, as the fluorescence signal gradually attenuates over time.

  • e.
    Analyze the data using FlowJo (FlowJo, USA) software.

    Note: During flow cytometry detection, we collect more than ten thousand events, with at least five thousand viable cells to ensure the accuracy of the experiment, as shown in the example of Figure S1.

Figure 2.

Gating strategy of lysotracker and AO staining

Flow cytometry gating strategy to measure lysosome quantity by lysotracker staining (A) and membrane integrity by AO staining (B). The fluorescence of lysotracker staining is quantified by employing a 638 nm laser with a 660/10 nm bandpass filter. For AO staining, the fluorescence is measured using a 488 nm laser with dual filters: a 525/40 nm (Green) and a 690/50 nm (Red) filter.

Acridine orange (AO) staining

Timing: 1 h 30 min

AO accumulates in intact lysosomes, emitting a bright red fluorescence under blue or green excitation light, and it emits green fluorescence once it is released from damaged lysosomes.³ In this step, the membrane integrity of lysosomes is examined.

Note: This step should be protected from exposure to light. Turn off the light or cover the tubes with a piece of aluminum foil paper.

• 5.
  Working solution preparation.

  • a.
    Quickly centrifuge the vial at 3000–4000 ×g for 30 s before opening the lid of Acridine Orange hydrochloride.
  • b.
    Reconstitute in 20 mL sterile DMSO to a concentration of 5 mg/mL.

    Note: Allow vortexing or sonication to ensure adequate dissolution.

    Note: AO stock solution (5 mg/mL) could be stored in working aliquots at −20°C to −80°C for more than three months.

  • c.
    Thaw the AO stock solution at 32°C.
  • d.
    Add AO stock solution (5 mg/mL) to the cell culture medium at a ratio of 1:10000 to achieve a final concentration of 0.5 μg/mL.
• 6.
  Harvesting of the cells.

  • a.
    Collect the cultured cells into a 15 mL centrifuge tube.
  • b.
    Centrifuge the 15 mL tube at 300 ×g for 5 min.
  • c.
    Discard the supernatant and resuspend these cells with 5 mL of PBS.
  • d.
    Centrifuge the 15 mL tube at 300 ×g for 5 min.
  • e.
    Discard the supernatant.
• 7.
  Staining with AO and DAPI.

  • a.
    Resuspend these cells with AO working solution to a concentration of 2 × 10⁵ cells per mL.
  • b.
    Add DAPI at a final concentration of 5 μg/mL.
  • c.
    Incubate the cells for 20 min at 37°C in the dark.
• 8.
  Sample collection and washing.

  • a.
    Collect the stained cells in a flow cytometry tube.
  • b.
    Pipette 1 mL of PBS into the flow cytometry tube for every 2 × 10⁵ cells.
  • c.
    Centrifuge the tube at 300 ×g for 5 min.
  • d.
    Discard the supernatant.
  • e.
    Repeat steps 8b–8d twice.
• 9.
  Measurement and analysis.

  • a.
    Resuspend these cells with 400 μL of PBS.
  • b.
    Measure fluorescence using a 488 nm laser with 525/40 (Green) filter and 690/50 (Red) filter on DxFLEX (Beckman Coulter). Flow cytometry gating strategy to obtain MFI of AO staining is shown in Figure 2B.

    Alternatives: Other flow cytometers, such as the FACS Canto II (BD Biosciences), can also be chosen for flow cytometric analysis.

  • c.
    Analyze the data using FlowJo software.

Construct a stable cell line expressing LGALS3-EGFP

Timing: 2 weeks

Note: Ensure the necessary BSL-2 biosafety level to work with lentiviruses.

The galectin puncta assay is one of the standard methods for detecting lysosomal membrane permeabilization (LMP). Galectin-3/LGALS3, in particular, is highly expressed in a variety of cell types, rapidly responds to damaged lysosomes, and is broadly applicable in numerous research applications.⁴ These attributes render LGALS3 a powerful tool for studying LMP and associated cellular processes. In this step, we establish cell lines expressing LGALS3-EGFP to detect LMP.

• 10.Plasmid preparation: Obtain the LGALS3-EGFP plasmid from IGEbio and prepare the plasmid with a concentration of more than 200 ng/μL with EndoFree Mini Plasmid Kit following the manufacturer’s manual: https://www.tiangen.com/upload/file/20230811/20230811140202_92266.pdf. The sequence information of the LGALS3-EGFP plasmid is provided in Data S1.

Alternatives: The LGALS3 plasmid used is also available via Addgene.

• 11.
  293T preparation.

  • a.
    Cell plating: Plate the 293T cells at a density that will result in approximately 5 million cells per 60 mm dish.
  • b.
    Incubate the cells at 37°C with 5% CO₂ overnight.
  • c.
    Assessment of cell condition: On the day of transfection, examine the cells for confluency.

Note: The ideal density for transfection is 70%–80%. If the cells have reached the appropriate density, proceed with the following steps.

• 12.
  Viral packaging.

  • a.
    Co-transfect the LGALS3-EGFP plasmid with psPAX2 and pMD2.G into 293T cells using Lipo3000 according to the manufacturer’s instructions: https://www.thermofisher.cn/document-connect/document-connect.html?url=https://assets.thermofisher.cn/TFS-Assets%2FLSG%2Fmanuals%2Flipofectamine3000_protocol_Chinese.pdf.

    Note: LGALS3-EGFP, psPAX2, and pMD2.G plasmid are used at a ratio of 8:6:4 μg.

  • b.
    Incubate the transfected 293T cells at 37°C with 5% CO₂ for 4–6 h.
  • c.
    Replace the transfection medium with fresh DMEM complete growth medium and incubate for an additional 48 h.
• 13.
  Virus harvesting and purification.

  • a.
    Collect the media containing the virus 48 h post-transfection.
  • b.
    Clarify the media with a 0.45 μm sterile syringe filter to remove cellular debris.
• 14.
  Infection of AML cells.

  • a.
    Ensure the MV4-11 cell line is in the logarithmic phase of growth, and plate at 1.0×10⁵ cells/well in a 12-well plate.
  • b.
    Add the LGALS3-EGFP lentivirus to the cell suspension at the optimized MOI.

    Note: To enhance infection efficiency, it's crucial to optimize the MOI for your specific cell type. For MV4-11 cell lines, we recommend an MOI range of 30–50.

  • c.
    Enhanced infection: Add polybrene (5 μg/mL) to cell suspension and mix gently.

    Note: Given the nature of suspension cells, it's highly recommended to add reagents that enhance lentiviral infection, such as polybrene. For adherent cells, this step is optional.

  • d.
    Centrifuge the plate: Carefully seal the cell culture plate, and centrifuge at 300 ×g for 60–90 min at 32°C.

    Note: For adherent cells, this step is not mandatory.

  • e.
    Incubation: Incubate the cells with the virus for 12 h in a cell culture incubator at 37°C with 5% CO₂.
• 15.
  Post-infection culture and selection.

  • a.
    After incubation, replace the medium to remove excess virus and polybrene, and continue to culture for 48–72 h for LGALS3-EGFP expression.
  • b.
    Selection: Add the selected antibiotic (e.g., puromycin) to the culture. For puromycin, 2 μg/mL is sufficient to select genetically modified MV4-11 cells.

    Note: The choice of antibiotic depends on the resistance gene carried by the backbone of the lentiviral vector. If the antibiotic resistance gene is not included, Fluorescence-Activated Cell Sorting (FACS) is an alternative method.

    Note: The concentration for selection of each cell line should be determined experimentally using a kill curve.

  • c.
    Maintenance of selection: Change the medium to fresh medium containing selection antibiotic regularly (every 2–3 days) for 1–2 weeks.
• 16.Validation: Use flow cytometry to confirm the stable expression of EGFP. Usually, an EGFP-positive rate of 95% is expected.

Note:Figure 3 illustrates the validation of LGALS3-EGFP expression in MV4-11 cells.

Figure 3.

Validation of LGALS3-EGFP expression in MV4-11 cells

(A) The constructed MV4-11 cells expressing LGALS3-EGFP are observed under an inverted fluorescence microscope by using a green fluorescence filter set. In this assay, the wild-type MV4-11 cells are employed as a negative control.

(B) The validation is conducted by measuring the fluorescence by using a 488 nm laser with 525/40 (Green) filters using DxFLEX (Beckman Coulter). In this assay, the wild-type MV4-11 cells are employed as a negative control.

Induce lysosomal damage

Timing: 2 days

• 17.
  Cell seeding and treatment application.

  • a.
    Plate cells in a 12-well plate at 1.0×10⁶ cells/well.
  • b.
    Add Ven and HA to the combo group and a corresponding volume of DMSO to the DMSO group as a control.
  • c.
    Mix the wells gently.
• 18.Incubate for 48 h at 37°C with 5% CO₂.
• 19.Harvest and wash the cells with 2 mL PBS twice.

Detection of LGALS3 puncta

Timing: 3–4 h

• 20.Fixation: Resuspend the cell pellet in the 4% PFA and incubate at 32°C for 30 min. Gently pipetting cells to ensure a single-cell suspension.
• 21.Wash: Remove 4% PFA by centrifugation and gently resuspend the cells in PBS.
• 22.Nuclear staining: Add DAPI at a final concentration of 5 μg/mL and incubate for 10 min at 32°C.
• 23.Wash: Remove the DAPI solution by centrifugation and gently resuspend the cells in 500 μL PBS.
• 24.Adherence to slides: Carefully drop the cell suspension onto the poly-L-lysine coated slides, and place the slide in the dark for 30 min to allow the cells to settle and adhere.
• 25.Mounting: Apply a drop of antifade mounting medium to the cell and place a coverslip over the cells carefully.
• 26.Seal the edges of the coverslip with nail polish.

Note: Avoid to create air bubbles.

• 27.Capture images using a Zeiss LSM880 Airyscan confocal microscope.

Expected outcomes

Figure 4 highlights data obtained using the above protocol. The data represent the expected results that the synergism of the HA and Ven combination leads to the accumulation of damaged lysosomes, which trigger cell death in AML cells.

Figure 4.

An example of the representative results obtained from this protocol

(A) Lysotracker staining in the DMSO and Combo (HA plus Ven) group. MV4-11 cells treated with DMSO or Combo for 48 h were labeled with Lysotracker Deep Red and analyzed by flow cytometry. MFI, mean fluorescent intensity.

(B) AO staining in the DMSO and Combo group. MV4-11 cells treated with DMSO or Combo for 48 h were labeled with AO and analyzed by flow cytometry. The red-to-green fluorescence intensity ratio was calculated by comparing the MFI of signals measured at 690/50 nm (Red) with that captured at 525/40 nm (Green).

(C–E) Immunofluorescence and confocal laser scanning microscopy. MV4-11 cells were transfected with a plasmid encoding LGALS3-EGFP and treated with DMSO or Combo. Treated cells were fixed and mounted on slides. Representative images (C and D) and quantification of cells with EGFP-LGALS3 puncta (E) are shown. Scale bar, 5 μm. All fields were imaged with confocal microscopy using a 63× objective. Data are presented as the mean ± SD, and differences were compared using Student’s t test. ∗, p < .05, ∗∗∗p < .001.

The result of lysotracker staining demonstrated the accumulation of lysosomes (Figure 4A) in the HA plus Ven group. As for AO staining, as expected, HA plus Ven treatment led to an increase in green fluorescence intensity coupled with a decrease in red fluorescence, indicating the occurrence of LMP (Figure 4B). The assay for galectin puncta was conducted using a confocal microscope to visualize EGFP-LGALS3 puncta. LGALS3 is a cytosolically synthesized lectin with a natural affinity for β-galactoside.⁵ In steady-state, LGALS3 is located in the cytosol and nucleus, showing a diffuse distribution under confocal microscopy (Figure 4C). In the lysosome-damage condition, β-galactosides are exposed on the cytosolic side of the lysosomal membrane, which leads to the recruitment of LGALS3 to the leakage.⁴ In this protocol, HA plus Ven induces severe lysosome rupture, and LGALS3 puncta is observed (Figures 4D and 4E).

Quantification and statistical analysis

For flow cytometry analysis of lysotracker staining and AO staining, the MFI was quantified using FlowJo software. To ensure the reliability of the results, three independent replicates were conducted, and intergroup comparisons were performed. To quantify LMP in galectin puncta assay, the number of cells displaying LGALS3 puncta was tallied to determine the proportion, and statistical comparisons were made across three distinct fields of view within each group. Statistical analysis was carried out using GraphPad Prism version 8.0. Data are presented as the mean ± standard deviation (SD). Student’s t test was employed to compare differences between the two groups. p < 0.05 was considered statistically significant. Data with statistical significance (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.001) are shown in Figures.

Limitations

Compared to adherent cells, the preparation of high-quality live cell monolayers from suspension cells, particularly blood cells of small size, presents a certain degree of difficulty when it comes to staining and observation. Lysosomal fluorescence assays offer a simplified and quantifiable approach to a certain extent. Nevertheless, the interpretation of these assays is susceptible to interference from cell viability, and the specificity of the staining can be compromised. In addition, the lysosomal fluorescence may constitute only a small portion of total cellular fluorescence in some experiments, making it difficult to quantify the number of lysosomes by flow cytometry. If drug treatment produces only very weak lysosomal accumulation, it would be difficult to observe a peak shift. In this case, the protocol of measuring the lysosomal fluorescence by using flow cytometry is not suitable. Besides, an increase in MFI of lysotracker dyes could be a result of any one of the following scenarios: 1) decrease of lysosomal pH; 2) larger number of lysosomes; 3) swollen lysosomes to increase the volume of the lysosomes. Therefore, in certain circumstances, real-time lysosomal monitoring based on live-cell imaging can more accurately and specifically reveal changes in lysosomal function and activity. Currently, some articles have demonstrated a variety of excellent methods for this kind of real-time monitoring, including AO monitoring of lysosomal membrane permeability changes, detection of lysosomal enzyme activity, and monitoring of lysosomal pH.^6,7,8

Additionally, AO is not a lysosome-specific dye, it also permeates the nuclear membrane, staining nuclear DNA and RNA, which results in a uniform green or yellow-green fluorescence in the cell nucleus. In apoptotic cells, due to chromatin condensation or fragmentation into varying sizes, AO staining can exhibit dense, intensely yellow-green fluorescence, whereas in necrotic cells, the yellow fluorescence is diminished or even disappears.^9,10 Therefore, when using AO to detect LMP, it is important to focus on the changes in the intensity of the dual fluorescence in the cytoplasm, or alternatively, select live cells for analysis.

This protocol provides a relatively simple strategy to evaluate lysosome alterations in suspension cells with small sizes. However, there are still alternative methods to be used. For example, immunofluorescence detection of lysosomal structural proteins, such as LAMP1, is a more accurate method to monitor the changes in lysosomal number.

Troubleshooting

Problem 1

Extensive cell death in the drug treatment group related to Step 4d.

Potential solution

• •CCK8 assay validation: Lysotracker products can stain live cells in acidic environments and are used to label and trace acidic organelles such as lysosomes. We recommend conducting the CCK8 assay following drug treatment to ensure approximately 50% cell viability, which would lend greater credibility to the experimental outcomes (Figure 5).
• •Add a LIVE/DEAD dye such as DAPI to further facilitate the gating of live cells.

Figure 5.

Utilization of the CCK8 assay to determine the optimal cells for detecting lysosomal alterations

Problem 2

MFI of Lysotracker Deep Red staining is too weak and related to Step 4d.

Potential solution

• •Stain in the dark and detect the fluorescent signal immediately after staining.
• •Extending the staining time to 1 h, or increasing the concentration of the working solution (75 nM).
• •For the possibility of treatment groups producing only very weak lysosomal accumulation, confocal detection is a better choice. You can stain and wash these cells as in this protocol. Subsequently, prepare slides and observe the cells using a confocal microscope fitted with the correct filter set. Here, we provide an example of images with weak fluorescence intensity captured using a Zeiss LSM880 Airyscan confocal microscope using a 63× objective in Figure 6A, which is challenging to observe and track the fluorescent puncta of acidic organelles in such slides. The MFI measured by flow cytometry at this point is also deemed unreliable.

Figure 6.

Lysotracker staining fields imaged with confocal microscopy

MV4-11 cells are treated with DMSO or Combo (HA plus Ven) for 48 h and observed by confocal microscope using a 63X objective.

(A) An example of weak fluorescence signal.

(B and C) The confocal microscopy photographic effects of non-fixed (B) and fixed (C) cells.

Problem 3

The inaccuracy in quantifying the number of lysosomes using flow cytometric examination of lysotracker staining at step 4.

Potential solution

• •Fix the cells before mounting and microscopy image acquisition for observation after lysotracker staining: In the manufacturer’s instructions, it is advised to perform direct fluorescence detection on live cells under a microscope, as the use of fixatives can diminish the intensity of fluorescence. However, the manufacturer’s protocol is not optimal for suspension cells. Direct observation of non-fixed live suspension cells is challenging to focus on and localize. Moreover, the slide preparation of suspended cells requires time, which delays observation. Additionally, the small size of the cells makes them difficult to observe, leading to poor photographic quality. Therefore, in the protocol described in our preceding section, we believe that the MFI of flow cytometry can reflect changes in the quantity of lysosomes as a starting point.

Based on our experiment experience and methods from other groups, although fixation results in loss of fluorescence intensity, the benefits it provides for suspended cells are greater.¹¹ Following staining with lysotracker dye for 30–60 min, cells were fixed with 4% paraformaldehyde for 20 min and then slides were prepared for observation according to steps 21 to 27.

Utilizing this method, the cell morphology becomes more amenable to focus, and fluorescence after fixation can be preserved for 2 days in the dark. Figures 6B and 6C respectively illustrate the confocal microscopy photographic effects of fixed and non-fixed cells, under the same treatment and laser intensity conditions.

• •Conducting alternative experiments: Western blots or immunofluorescence of key lysosome proteins such as LAMP1.
• •Directly observe the quantity and morphology of lysosomes under a transmission electron microscope.

Problem 4

Few cells in one view under confocal microscopy at step 27.

Potential solution

Cells can be lost during each centrifuge or slide adhesion. Use a swinging-bucket rotor centrifuge to minimize cell loss. As well, use slides coated with poly-L-lysine instead of ordinary glass slides to enhance cell adhesion.

Problem 5

Abnormal cell morphology at step 27.

Potential solution

DAPI staining helps to assess the localization and status of cells. Nuclei with condensed, fragmented, or irregular shapes indicate apoptotic status. These cells should be excluded from the assessment. Lysosome damage detection should focus on cells with normal nuclei, which are round and oval-shaped (Figure 7).

Figure 7.

Nuclear morphology of cells stained with DAPI

The morphology of nuclei in MV4-11 cells treated with HA and Ven was observed. Nuclei enclosed by a red dashed square frame indicate normal morphology, while yellow dashed square frames exhibit nuclei with condensed, fragmented, or irregular shapes.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Hui Zeng (androps2011@hotmail.com).

Technical contact

Technical questions on executing this protocol should be directed to and will be answered by the technical contacts, Kexiu Huang (huang@stu2022.jnu.edu.cn).

Materials availability

This study did not generate new unique reagents.

Data and code availability

None.

Acknowledgments

The authors would like to thank our funding sources, including the National Natural Science Foundation of China (grant no. 81970143, no. 82270167), the Talent Young Program of Guangdong Province (2021B1515020017), and the Leading Talents Program from The First Affiliated Hospital of Jinan University to H.Z.

Author contributions

H.Z. designed the project; X.J. and K.H. performed most experiments; K.H., X.J., J.D., and H.Z. analyzed the data; K.H., J.D., and H.Z. wrote and revised the manuscript. All authors have read and approved the final submitted manuscript.

Declaration of interests

The authors declare no competing interests.

Declaration of generative AI and AI-assisted technologies in the writing process

During the preparation of this work, we used Grammarly in order to correct grammatical mistakes. After using this tool, we reviewed and edited the content as needed and take full responsibility for the content of the publication.