Lysosomal membrane damage in HaCaT cells was determined using the acidotropic probe LysoTracker Blue DND-22 (Invitrogen by Thermo Fisher Scientific, Waltham, MA, USA) whose fluorescence depends on pH. HaCaT cells were incubated with 400 nM LysoTracker Blue DND-22 in PBS-G, then washed twice with PBS-G, and subsequently irradiated with UVB for 6 min. Due to the strong photobleaching effect of this probe caused by UVA radiation, the cells were not exposed to UVA radiation (Figure S1 ). The fluorescence intensity of LysoTracker Blue DND-22 with an excitation wavelength of 380 nm and an emission wavelength of 422 nm was measured using the Tecan Infinite 200pro fluorescence reader.
Lysotracker blue dnd 22
Manufactured by Thermo Fisher Scientific
Sourced in United States
LysoTracker Blue DND-22 is a fluorescent probe used for labeling and tracking acidic organelles, such as lysosomes, in living cells. It selectively accumulates in these organelles due to their low pH environment.
Automatically generated - may contain errors
Lab products found in correlation
Zen blue software, by Zeiss (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Trypsin, by Atlanta Biologicals (2 mentions)
Poly l lysine (pll), by Merck Group (2 mentions)
Infinite 200 pro, by Tecan (1 mentions)
Sp8 confocal microscope, by Leica (1 mentions)
Cellmask deep red plasma membrane stain, by Thermo Fisher Scientific (1 mentions)
Axiovision 4, by Zeiss (1 mentions)
Mitotracker red cmxros, by Thermo Fisher Scientific (1 mentions)
Fv1000 confocal microscope, by Olympus (1 mentions)
Cellmask deep red, by Thermo Fisher Scientific (1 mentions)
Sp8 confocal microscopy, by Leica (1 mentions)
Fugene hd transfection reagent, by Promega (1 mentions)
Spinsr10, by Olympus (1 mentions)
D141400, by Matsunami (1 mentions)
Bafa1, by Merck Group (1 mentions)
Pi 4 5 p2 dic8, by Echelon Biosciences (1 mentions)
Chloroquine, by Merck Group (1 mentions)
Apilimod, by MedChemExpress (1 mentions)
Oregon green 488 dextran, by Thermo Fisher Scientific (1 mentions)
36 protocols using lysotracker blue dnd 22
1
Lysosomal Damage Measurement in HaCaT Cells
2
Visualizing CpG-C Uptake in Cells and Tissues
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Cultures of N9 cells grown on coverslips were treated with CpG-C-TAMRA for 24 hours and washed three times. LysoTracker Blue DND-22 (50 nM, Thermo Fisher Scientific) was applied for 30 minutes at 37°C, and coverslips were washed and mounted on slides. For staining of CpG-C uptake in vivo, a single cell suspension was prepared from CX3CR1GFP/+ mice treated with CpG-C-TAMRA as described in the ImageStream FACS analysis protocol herein. LysoTracker Blue DND-22 (50 nM, Thermo Fisher Scientific) was then mixed into the cell suspension for 30 minutes at 37°C, and cells were mounted on a cover glass and imaged with a Leica SP8 confocal microscope using a ×63 (NA– 1.4) oil immersion objective (Fig 5B ). Similarly, for the cells extracted from the brains of animals, we imaged only green fluorescent protein (GFP)–positive cells (i.e., microglia).
3
Quantifying MAP Invasion and Phagosome Acidification
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MDMs and MAC-T cells were separately seeded at 2 × 104 cells/well in a 24 well plate containing No. 1.5 glass cover slips. MAP invasion using MAP K-10(pWes4)-GFP was conducted using the same conditions described above with the exception of an 1 h pre-incubation step with 50 nM of bafilomcyin A1 (A.G. Scientific Inc., San Diego, CA). Phagosome acidification was determined by a LysoTracker staining method (Lamont et al., 2012 (link)). Upon the final 30 min of each post-infection time point, 25 nM of LysoTracker Blue DND-22 (Invitrogen, Carlsbad, CA) was added to cell medium. Cells were subsequently washed thrice in Dulbecco's PBS, incubated in pre-warmed Deep Red CellMask plasma membrane stain (2.5 μg/mL) (Invitrogen, Carlsbad, CA) for 5 min, and rewashed. Cells were fixed in absolute methanol for 5 min at −20°C and washed twice in ice-cold D-PBS. Slides were stored at 4°C until visualization by confocal microscopy. An Olympus FluoView 1000 upright confocal microscope (Olympus, South-end-on-sea, Essex, United Kingdom) was used to image infected and control cell slides slides with FITC, Cy5, and DAPI lasers. Z-series for each slide was taken in 1 μM steps and stacked to render a complete image. A minimum of three fields per slide were imaged. Blocking assay was repeated in triplicate for each condition.
4
Colocalization of Particles and EGFR
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Colocalization of the exposed particles with lysosomes and EGFR was evaluated after 6 h and 24 h. Following exposure to EGF/particles, cells were washed three times with PBS before staining with a solution containing 100 nM LysoTracker Blue DND-22 (Invitrogen, Thermo Fisher Scientific Inc., Switzerland) in cRPMI for 30 min. Before cell imaging, samples were washed twice with PBS and phenol-free cRPMI was added. Z-stacks were acquired sequentially, collected with a field of view of 144.80 μm x 144.80 μm with a pixel density of 1880 × 1880 and a pixel dwell time of 0.31 μs. Data are shown as mean + standard error of the mean for n = 10 cells. The Pearson’s correlation coefficient (PCC) was determined using EzColocalization (Image J plugin).28 (link)
5
Quantifying Mitophagy in AR42J Cells
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After GFP-RFP-pMITO transfection, treated AR42J cells were observed by the inverted confocal microscope Olympus FV1000 (PLAPON/1.42) to quantify mitophagy. The area of mitochondria per cell localized in autolysosomes (RFP-MITO) was quantified using the Fiji-win64 software.
To analyze mitochondria and lysosomes localization, treated AR42J cells were incubated with 200 nM of MitoTracker Red CMXRos and 50 nM of LysoTracker Blue DND-22 (Invitrogen) for 30 min at 37°C and subsequently washing them three times with PBS. Cells images were acquired through an Olympus Confocal Microscope FV1000 and images were acquired using the Zen Blue software (Zeiss) and processed on the AxioVision 4.2 software (Carl Zeiss). To analyze lysosomal area per cell, treated AR42J cells labeled with LysoTracker were analyzed in an Olympus Confocal Microscope FV1000 and quantified using the Fiji-win64 software in arbitrary units.
To analyze mitochondria and lysosomes localization, treated AR42J cells were incubated with 200 nM of MitoTracker Red CMXRos and 50 nM of LysoTracker Blue DND-22 (Invitrogen) for 30 min at 37°C and subsequently washing them three times with PBS. Cells images were acquired through an Olympus Confocal Microscope FV1000 and images were acquired using the Zen Blue software (Zeiss) and processed on the AxioVision 4.2 software (Carl Zeiss). To analyze lysosomal area per cell, treated AR42J cells labeled with LysoTracker were analyzed in an Olympus Confocal Microscope FV1000 and quantified using the Fiji-win64 software in arbitrary units.
6
Visualizing Lysosome Dynamics in SL2 Cells
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SL2 cells were seeded (2 × 105 cells per dish) in 35 mm dish with glass bottom one day before infection. Cells were infected with amastigotes and at determined times, cell membranes were stained with CellMask deepRed for 10 min, and acidic organelles were stained with Lysotracker Blue DND-22 (Invitrogen). Confocal images were taken using a Leica SP8 confocal microscopy using a 63x objective. Images were edited using Fiji software (Schindelin et al., 2012 (link)).
7
Intracellular Delivery of PIP3 in αSyn Cells
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Intracellular delivery of PIP3 was performed using the PIP3 Shuttle PIP™ Kit (P-9039, Echelon Biosciences), with slight modifications to the manufacturer’s protocol. Briefly, HeLa cells stably overexpressing αSyn-mRFP were seeded on a 4-well glass bottom dish (Matsunami, D141400) and incubated overnight at 37 °C. Shuttle PIP carrier 2 (Histone H1) and Bodipy®-FL-PIP3 were incubated in a 0.2 ml tube in a 1:1 molar ratio for 10 min at room temperature. The complex was diluted with Opti-MEM and added to media covering HeLa cells with a final carrier and PIP3 concentration of 5 µM. The following day, the dye-containing media was removed, and cells were washed with phosphate-buffered saline (PBS) before live imaging using SpinSR10 (Olympus) or fixation with 4% paraformaldehyde (PFA) for immunostaining. Confocal images were taken randomly across the entire well at 60× magnification, and the percentage of αSyn puncta-positive cells was quantified. For transient gene overexpression, HeLa cells were seeded and transfected with pcDNA-αSyn-mRFP 24 h prior to delivery of PIP3 using Fugene HD transfection reagent (E2311, Promega Corporation), according to the manufacturer’s protocol. For live imaging of lysosomes, cells were incubated with 300 nM LysoTracker™ Blue DND-22 (L7525, Invitrogen) for 1 h and washed three times with PBS before observation.
8
Lysosomal Trafficking and Acidity Assay
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Apilimod (MedChemExpress); WX8 (gift from Juan S. Bonifacino lab) Oregon Green 488-dextran 10,000 mW (OG; Thermo Fisher); chloroquine, BafA1, monensin, and nigericin (Sigma-Aldrich); LysoTracker Blue DND-22 (Invitrogen); Magic Red cathepsin B essay (ImmunoChemistry Technologies); PI(3,5)P2 diC8, PI(4,5)P2 diC8, PI3P diC8 (Echelon Biosciences).
9
Chitosan-based Nanoparticles for Targeted Delivery
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Chitosan with an approximate 15.0 kDa average molecular weight was obtained by enzymatic degradation of 95% deacetylate chitosan (Mw = 450 kDa) and was supplied by Yuhuan Marine (Yuhuan, China). Stearic acid (SA) was purchased from Fluka (Milwaukee, WI, USA). 3, 3′-dithiodipropionic acid was purchased from Tokyo Chemical Industry (Tokyo, Japan). FAM-siRNA (FAM-5'-UUCUCCGAACGUGUCACGUTT-3), Non-coding siRNA (siNC, 5′-UUCUUCGAACGUGUCACGUTT-3), and Bcl-2 specific siRNA (siBcl-2, 5′-CCCUGUGGAUGACUGAGUATT-3) was purchased from Shanghai GenePharma (Shanghai, China). Paclitaxel (PTX) was purchased from Shanghai Zhongxi Sunve (Shanghai, China). Cell Cycle Detection Kit was obtained from Keygene Biotech (Nanjing, China). L-Glutathione (GSH) and Nile red (NR) were purchased from Sigma-Aldrich (Diegem, Belgium). Fluorescein isothiocyanate (FITC), Rhodamine B Isothiocyanate (RITC), and buthionine sulfoximine (BSO) were purchased from Sigma (St. Louis, MO, USA). Cy5 labeled GAPDH molecular beacon (Cy5-GAPDH-MB, 5′-Cy5-CGACGGAGTCCTTCCACGATACCACGTCG-Dabcyl-3′) was purchased from Sangon Biotech (Shanghai, China). Lipofectamine™ 2000 and LysoTracker®Blue DND.22 was purchased from Invitrogen (Carlsbad, US). All of the other chemicals were of analytical or chromatographic grade.
10
Visualizing NMJs and Lysosomes in Drosophila
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In the visualization of neuromuscular junctions (NMJs), third instar larvae were dissected in HL3 saline 19 (link) and then fixed in 4% paraformaldehyde/PBS for 30 min. The blocking solution contained 2% bovine serum albumin and 0.1% Triton X-100 in PBS. FITC-conjugated goat anti-horseradish peroxidase (HRP) (1 : 1000; MP Biochemicals, Tokyo, Japan) was used as the detection antibody. Samples were mounted in Vectashield (Vector Laboratories; Burlingame, California, USA) and inspected under a confocal laser scanning microscope (Olympus FluoView FV10i; Olympus, Tokyo, Japan). Motoneurons in muscle 4 in abdominal segment 2 were inspected. The Meta Morph Imaging System 7.7 (Molecular Devices Inc., San Jose, California, USA) was used to measure nerve terminal branch lengths and Ib bouton sizes.
In the visualization of lysosomes, fat bodies were collected by dissecting third instar larvae in PBS and stained with 100 nM of LysoTracker Blue DND22 (Invitrogen, Tokyo, Japan) for 1 min. Stained samples were washed twice with PBS, mounted in Vectashield (Vector Laboratories, Burlingame, California, USA), and observed under a confocal laser scanning microscope (Olympus FluoView FV10iOlympus, Tokyo, Japan). The Meta Morph Imaging System 7.7 (Molecular Devices Inc.San Jose, California, USA) was used to measure the diameters of lysosomes.
In the visualization of lysosomes, fat bodies were collected by dissecting third instar larvae in PBS and stained with 100 nM of LysoTracker Blue DND22 (Invitrogen, Tokyo, Japan) for 1 min. Stained samples were washed twice with PBS, mounted in Vectashield (Vector Laboratories, Burlingame, California, USA), and observed under a confocal laser scanning microscope (Olympus FluoView FV10iOlympus, Tokyo, Japan). The Meta Morph Imaging System 7.7 (Molecular Devices Inc.San Jose, California, USA) was used to measure the diameters of lysosomes.
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