Endocytosis of vesicle into cytoplasia results in the formation of intracellular vesicles. Permeabilization of vesicle activates various signal pathways. Chromophore-assisted laser inactivation is used to study the consequence of intracellular vesicle rupture.
A spatial and temporal control of subcellular damage to intracellular vesicles can be achieved by adjusting the standing condition and light illumination. To begin, maintain HeLa cell culture in DMEM supplemented with 10%FBS. After washing the cells with PBS, Trypsinize the cells.
Then resuspend the cells in DMEM supplemented with 10%FBS. Dilute 300 nanograms of Gal3-GFP plasmid in 25 microliters of reduced serum medium. Then add 0.6 microliters P3000 reagent.
And gently mix, followed by vortexing the solution. Dilute 0.45 microliters of Lipofectamine 3000 reagent in 25 microliters of reduced serum medium and gently mix. Incubate for five minutes at room temperature.
Then mix diluted DNA and diluted Lipofectamine 3000 and incubate for 10 minutes at room temperature. After mixing the DNA lipofectamine mixture, 300, 000 suspended HeLa cells and 200 microliters of DMEM with 10%FBS, seed the cells on the central glass region of a 35-millimeter glass button dish. To allow cell attachment, incubate the cells at 37 degrees Celsius in the incubator supplied with 5%carbon dioxide for at least four hours.
Dilute AlPcS2a in DMEM supplemented with 10%FBS to make a one micromolar AlPcS2a solution. Prewarm the AlPcS2a containing-medium in a 37 degrees Celsius water bath and replace the culture medium with the AlPcS2a-containing medium. Incubate the cells at 37 degrees Celsius in the incubator supplied with 5%carbon dioxide overnight.
The following day, wash the cells twice with PBS to remove extracellular AlPcS2a after replacing the medium with fresh medium. Incubate at 37 degrees Celsius for four hours to allow residual dye along the endocytic pathway to accumulate into lysosomes. To manipulate single cells by damaging lysosomes, place the culture dish on the stage of a confocal microscope.
Use the 488 nanometer and 561 nanometer lasers for exciting GFP and AlPcS2a, respectively. Circle a five-by-five micrometer square region of interest on the AlPcS2a labeled vesicles which are selected to be damaged. Then pulse illuminate the region of interest with a 633 nanometer laser of 0.21 milliwatt, or 70 repeats.
Monitor the formation of Gal3 puncta as an indicator of lysosomal membrane permeabilization. Lysosomal staining with AlPcS2a in HeLa cells was performed using commercially-available markers, and were positively stained with green fluorescent dye. Lysosomes in TagRFP-Galectin3 expressed HeLa cells were stained with AlPcS2a.
Followed by focusing illumination with near infrared light, the results indicate that AlPcS2a-based chromophore-assisted laser inactivation was able to control local lysosomal rupture within the region of interest, leaving the rest of the lysosomes intact. This step is important because the incubation time will determine standing endosomes or lysosomes. For example, people can determine the size of by the release of membrane-impermeable dye.
Also can use pH sensitive dyes such as FITC dextran to difference endosomes and lysosomes. This technique is useful starting the downstream effects of intracellular vesicle membrane rupture. And there are downstream effects under various situations.
