1. AlPcS_2a stock preparation

1. Dissolve 10 mg of AlPcS_2a in 400 µL of 0.1 M NaOH. To improve solubility, heat the solution at 50 °C and vortex.
2. Mix the solution with 4 mL of phosphate-buffered saline (PBS). Then, filter the solution with a 0.22 µm filter to remove the insoluble precipitates.
3. Measure the concentration of the solution through a UV-Vis spectrophotometer. The extinction coefficient of AlPcS_2a at 672 nm is 4 x 10⁴ cm^-1 M^-1. Dilute the AlPcS_2a solution with PBS to make a 1 mM solution. Make 1 mL aliquots and store at -20 °C.

2. Transfection

NOTE: Gal3-GFP is applied as an indicator for live-cell imaging of lysosomal rupture.

1. Maintain HeLa cell culture in DMEM supplemented with 10% Fetal Bovine Serum (FBS). After washing the cells with PBS, trypsinize the cells, then resuspend the cells in DMEM supplemented with 10% FBS.
   NOTE: The method is applicable for most of the attached cell lines, including HeLa and A549 cell lines. In principle, despite not testing suspension cells, CALI assays could be performed with them.
2. Dilute 300 ng of Gal3-GFP plasmid in 25 µL of reduced serum medium, and then add 0.6 µL of P3000 reagent. Gently mix. Dilute 0.45 µL of Lipofectamine 3000 reagent in 25 µL of reduced serum medium. Gently mix.
3. Incubate for 5 min at room temperature. Mix diluted DNA and diluted Lipofectamine 3000, and then incubate for 10 min at room temperature.
4. Mix the DNA-Lipofectamine mixture, 3 x 10⁵ suspended HeLa cells, and 200 µL of DMEM+10% FBS, and then seed the cells on the central glass region of a 35 mm glass button dish.

3. AlPcS2a staining

1. To allow cell attachment, incubate the cells at 37 °C in the incubator supplied with 5% CO₂ for at least 4 h.
2. Dilute AlPcS_2a in DMEM supplemented with 10% FBS to make a 1 µM AlPcS_2a solution. Pre-warm the AlPcS_2a-containing medium in a 37 °C water bath. Replace the culture medium with the AlPcS_2a-containing medium.
3. Incubate the cells at 37 °C in the incubator supplied with 5% CO₂ overnight (16-18 h).
4. The following day, wash the cells twice with PBS to remove extracellular AlPcS2a. Replace the medium with fresh medium, and then incubate at 37 °C for 4 h to allow residual dye along the endocytic pathway to accumulate into lysosomes.
   ​NOTE: To stain endosomes, incubate cells in 1 µM AlPcS_2a in DMEM containing 10% FBS for 15 min at 37 °C. Next, wash cells twice with PBS and incubate them in pre-warmed medium before imaging .

Figure 1. A schematic figure representing the selective IV damage with AlPcS_2a. The figure shows the schematics of selective IV damage. Please click here to view a larger version of this figure.

Figure 2. Lysosomal staining with AlPcS2a. Lysosomes labeled overnight with 1 µM AlPcS_2a in HeLa cells are positively stained with 50 nM green fluorescent dye. Scale bar: 10 µm. Please click here to view a larger version of this figure.

4. Sample imaging and light illumination

NOTE: IV damage within a subcellular region of a single cell or all the AlPcS_2a-labeled cells in a culture dish can be performed. Bulk illumination of the whole culture dish allows quantitative study of this damage, including biochemical studies.

1. Manipulate single cells by damaging lysosomes within a single cell, as described below.
   1. Place the culture dish on the stage of a confocal microscope. Use the 488 nm and 561 nm lasers for exciting GFP and AlPcS2a, respectively.
   2. Circle a 5 x 5 µm² region of interest (ROI) on the AlPcS_2a-labeled vesicles which are selected to be damaged.
   3. Pulse illuminate the ROI with a 633 nm laser of 0.21 mW for 70 repeats. Monitor the formation of Gal3 puncta as an indicator of lysosomal membrane permeabilization.
2. Damage all labeled cells in a culture dish as described below.
   1. Place the culture dish on a platform. Illuminate the dish with 660 nm of collimated LED light (near-infrared light is ideal base on the excitation spectrum of AlPcS2a).
   2. Place the culture dish on the microscope and monitor the indicators of membrane permeabilization as mentioned in step 4.1.3.
3. Assess injury of IVs using the following indicators.
   1. The contents of endosome or lysosome are most highly glycosylated, which are exposed to cytosol upon IV rupture. Rapidly label these using cytosolic glycan-binding galectins, including galectin-1, galectin-3, galectin-8, and galectin-9^16,20.
   2. The size of the wound can be determined by the release of membrane-impermeable dye as described in²¹.
   3. The difference between endosomes and lysosomes are their luminal pH value. Lysosomal rupture perturbs the luminal pH, which can be measured by using pH-sensitive dye, such as FITC-dextran³.

Figure 3. AlPcS_2a-mediated CALI induces local recruitment of TagRFP-galectin-3 (Gal3) to the lysosomes within the illumination region. Lysosomes in TagRFP-galectin-3-expressed HeLa cells were stained overnight with 1 µM AlPcS_2a, followed by focusing illumination with near-infrared light (633 nm) within the yellow square. AlPcS_2a signal within the yellow square is photobleached, accompanied by the formation of TagRFP-galectin-3 puncta. Scale bar: 10 µm. Please click here to view a larger version of this figure.