Fetal bovine serum (FBS) was purchased from AusGeneX (Molendinar, Australia, CAT NO. FBS500-S); DMEM basic (1X) was from Gibco Life Technologies (Shanghai, China); penicillin/streptomycin solution (100 U/mL and 100 mg/mL, respectively), 2′, 7′-dichlorodihydrofluorescein diacetate (DCFH-DA), and Cellular grade DMSO were from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China); 3-(4,5-dimethyl-1,3-thiazol-2-yl)-2,5-diphenyl-2H-tetrazol-3-ium bromide(MTT) was obtained from Biosharp Life Technology Co., Ltd. (Hefei, China); Hoechest 33342, Mito-Tracker Green, and Lyso-Tracker Green were from Beyotime Biotechnology Co., Ltd. (Shanghai, China). All cells were cultured in a CO2 incubator (Wiggens, WCI-180, Beijing, China). All the in vitro fluorescence images experiments were obtained by using an Olympus IX73 + DP73 inverted microscope and laser confocal microscope (Carl Zeiss LSM 900, Jena, Germany). Hypoxic condition was created by coverslips (Citoglas, Hong Kong, China) and a hypoxia chamber (Stemcell Technologies, Cat 27310, Kent, WA, USA). Cytotoxicity assay was performed using an LED light (F&V, Z96kit, Fuijan, China) as irradiation source.
Lysotracker green
Manufactured by Beyotime
Sourced in China, United States
LysoTracker Green is a fluorescent probe that specifically labels acidic organelles, such as lysosomes, in living cells. It is a membrane-permeable dye that accumulates in these compartments due to their low pH environment. LysoTracker Green can be used to study the dynamics and distribution of lysosomes within cells.
Automatically generated - may contain errors
Lab products found in correlation
Hoechst 33342, by Beyotime (7 mentions)
Mito tracker green, by Beyotime (6 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (4 mentions)
Lsm 880, by Zeiss (4 mentions)
Mitotracker red, by Thermo Fisher Scientific (4 mentions)
Hoechst 33258, by Merck Group (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Mitotracker red, by Beyotime (2 mentions)
Golgi tracker green, by Beyotime (2 mentions)
Er tracker green, by Beyotime (2 mentions)
Fetal bovine serum (fbs), by Procell (2 mentions)
Hoechst 33342, by Solarbio (2 mentions)
Milli q plus system, by Merck Group (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Reactive oxygen species assay kit, by Beyotime (2 mentions)
Lsm 880 confocal microscope, by Zeiss (2 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
47 protocols using lysotracker green
1
Cytotoxicity Evaluation of Cells under Hypoxia
2
Staining and Imaging of Fat Droplets and Mitophagy in ZFL Cells
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To observe fat droplets in ZFL, the BODIPY staining was conducted as described in our previous study [25 (link)]. To assess the mitophagy state of ZFL, cells were plated on μ-slide 8 well (ibidi, Gräfelfing, Germany). Before treatment, ZFL was incubated in 100 nM Mtphagy Dye (Dojindo, Japan) and washed twice with the medium without FBS. Then, 5 μg/mL Hoechst 33342 (Invitrogen, Carlsbad, CA, USA) was used to stain the nuclei for 10 min at room temperature. To assess the co-localization of mitochondria and lysosomes, cells were stained by 300 nM Mitotracker Red (Invitrogen, USA) and 75 nM Lyso-Tracker Green (Beyotime, Shanghai, China) for 20 min, respectively. Fluorescence imaging was performed and photographed using a Leica sp8 laser scanning confocal microscope.
3
Characterization of Mitophagy Flux
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Lysosomes and mitochondria were labeled with the Lyso-Tracker Green (C1048; Beyotime, Shanghai, China) and Mito-Tracker Red (C1048; Beyotime, Shanghai, China) probes, respectively, and the co-localization of lysosomes and mitochondria was visualized to characterize mitophagy flux. Briefly, Lyso-Tracker Red solution was diluted into a final concentration of 50 nM using medium (1:13,333). Mito-Tracker Red solution was diluted into a final concentration of 20 nM using a medium (1:5000). Next, cells were incubated with Lyso-Tracker Red for 5 min at 37 °C followed by incubation with Mito-Tracker Red for 15 min at 37 °C. Finally, cells were observed with a fluorescence microscope.
4
Doxorubicin-Loaded Nanomedicine Synthesis
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Doxorubicin hydrochloride (DOX⋅HCl) was purchased from Beijing HuaFeng United Technology Co., Ltd (Beijing, People’s Republic of China). PEG (Mn: 2,000 g/mole), N,N′-carbonyldiimidazole,3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) and Hoechst 33258 were purchased from Sigma (St Louis, MO, USA). Roswell Park Memorial Institute 1640 without folic acid and fetal bovine serum were purchased from Thermo Fisher Scientific (Waltham, MA, USA). LysoTracker Green was purchased from Beyotime® Biotechnology Co., Ltd (Nantong, People’s Republic of China). Pluronic® F127 and Pluronic® P123 were kindly supplied by BASF Ltd. (Shanghai, People’s Republic of China). Purified deionized water was prepared by the Milli-Q plus system (EMD Millipore, Billerica, MA, USA). All the other reagents and chemicals were of analytical or chromatographic grade and were purchased from Concord Technology (Tianjin, People’s Republic of China).
5
Live-cell Imaging of Autophagy Flux
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For live-cell imaging, HEK293T cells overexpressing GLA wildtype or mutants were pre-transfected with pCS2-mCherry-LC3 or pCS2-mCherry-GFP-LC3 plasmid for 24 h and subsequently transferred to a μ-Dish (Φ 35 mm, NEST). For pCS2-mCherry-LC3 transfection, after 12 h of cell attachment, the cells were washed once with PBS, and 2 mL of serum-free medium supplemented with 50 nM LysoTracker Green (Beyotime, Shanghai, China, C1047S), a green fluorescent probe that can be selectively retained in lysosomes, was added. After coincubation for 30 min in the cell incubator, the medium was replaced with fresh medium without LysoTracker Green to avoid nonspecific labeling caused by an excessively long staining time. Confocal images of the cells were taken immediately with a laser scanning microscope (Zeiss, Oberkochen, Germany). The flux of autophagy in cells can be measured by the colocalization of LC3 with lysosomes. The excitation wavelength of LysoTracker Green was 488 nm, and the emission wavelength was 500–550 nm. mCherry-LC3 was excited at 561 nm and emitted from 570 to 620 nm. Images were processed using ImageJ 1.52a.
6
Fluorescent Probes for Oxidative Stress
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Tetraethyl orthosilicate (TEOS), triethanolamine (TEAH), coumarin (C9H6O2, 99%), 9,10-anthracenediyl-bis(methylene) dimalonic acid (ABDA), sodium dithionite (SDT) and decahydronaphthalene (98%) were purchased from Sigma-Aldrich. Potassium permanganate (97%) was obtained from Shanghai Chemical Co. Dichloromethane (99.99%) was purchased from Aladdin Industrial Inc. Dodecacarbonyltriiron (Fe3(CO)12, 96%) was purchased from Macklin Industrial Inc. All chemicals were used as received without any further purification. Hemoglobin (Hb) of bovine red blood cells was purchased from MP Biomedicals. DAPI, Lyso-Tracker Green, 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) and 2-[6-(4,-hydroxy)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid (HPF) were purchased from Beyotime Biotechnology.
7
Cellular Localization of Ce6-DOC@TA-Fe3+ Complex
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CT26 cells were seeded onto confocal dishes and cultured overnight. A fresh medium containing Ce6-doped DOC@TA-Fe3+ was added and incubated for 2, 4, 6, and 8 h. The cells were stained with Hoechst 33342 and Lysotracker green (Beyotime, Shanghai, China) for 10 and 20 min, respectively. Finally, colocalization was investigated via confocal laser scanning microscopy (CLSM; Zeiss LSM880, Oberkochen, Baden-Wurttemberg, Germany). The methods of cell culture, cell uptake, and cell cycle assay are shown in the Supplementary data.
8
Fluorescent Nanoparticle-Based Cellular Assays
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Fluorescent (Ex 620, Em 680 nm) polystyrene nanoparticles (PS) with a theoretical diameter of 100 nm were purchased from Tianjin BaseLine ChromTech Research Centre (Tianjin, China). Streptozotocin (STZ) and Hank's balanced salt solution (HBSS) were purchased from Solarbio Life Sciences (Beijing, China). TNBS (2, 4, 6-trinitrobenzene sulfonic acid), amiloride, chlorpromazine, and lovastatin were purchased from Sigma–Aldrich (St. Louis, MO, USA). Glycine sarcosine (Gly-Sar) was purchased from Tokyo Chemical Industry Co., Led. (Tokyo, Japan). Ezetimibe was purchased from Huaxia Chemical Reagents Co., Ltd (Chengdu, China). BCA protein assay kit, LDH assay kit, Reactive oxygen species (ROS) assay kit, 3,3′-dioctadecyloxacarbocyanine perchlorate (DiO), 4ʹ,6-diamidino-2-phenylindole (DAPI), Lyso-Tracker Green, ER-Tracker Green, Golgi-Tracker Green were purchased from Beyotime Biotechnology (Shanghai, China). Fluo-4 AM was purchased from Meilun Biotechnology (Dalian, China). Rabbit anti-rab5, mouse anti-rab7, rabbit anti-rab11, rabbit anti-claudin-1, and Alexa Fluor 488 labeled goat anti-rabbit/mouse IgG were purchased from Abcam (Cambridge, UK). Anti-VAMP8 antibody and anti-Annexin A2 antibody were purchased from HUABIO (Hangzhou, China). All other chemical reagents in this study were analytic grade.
9
Gambogic acid-mediated XIAP silencing in apoptosis
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Gambogic acid was purchased from Sigma-Aldrich (St. Louis, MO, USA). Cy3-labeled siRNA (XIAP siRNA) was purchased from RiboBio (Guangzhou, China) with the following sequences: XIAP siRNA (sense): 5-AAG UGG UAG UCC UGU UUC AGC-3. Monoclonal antibodies (XIAP) and HRP-linked antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Cell Count Kit-8, 4′,6-diamidino-2-phenylindole (DPAI), and LysoTracker Green were from the Beyotime Institute of Biotechnology (Shanghai, China). Annexin V-FITC Apoptosis Detection Kit and Cell Cycle and Apoptosis Detection Kit were from Beijing Biotopped Technology Co., Ltd. (Beijing, China). Tween-80, squalene, and glycerol were manufactured by Shanghai Energy Chemical Co., Ltd. Immunohistochemistry Kit (D601037-0050) was purchased from Sangon Biotech (Shanghai, China). Deionized water was produced in the laboratory. The reagent-grade chemicals used in the tests were all used exactly as provided to the researchers.
10
Multifunctional Nanoparticle Drug Delivery
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1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N [amin (polyethylene glycol)-2000] (DSPE-PEG2000-NH2) and Egg phosphatidylcholine (EPC) were obtained from AVT Pharmaceutical Technology Co., Ltd. (Shanghai, China). Lyso-Tracker Green was from Beyotime Biotechnology Co., Ltd. (Shanghai, China). Single-chain variable fragment (scFv) was provided by Merry Bio Co., Ltd. (Nanjing, China). Cholesterol, Hoechst33258, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Agarose and 4,6-diamino-2-phenylindole (DAPI) were acquired from Solarbio (Beijing, China). Nine arginine (R9) peptide was synthesized from Shanghai Taopu Biotechnology Co., Ltd. (Shanghai, China). DSPE-PEG-cRGD was synthesized by Xi’an Ruixi Biological Technology Co., Ltd. (Xi’an, China). Irinotecan base (IRI), Doxorubicin Hydrochloride (DOX·HCl) were purchased from Meilun Biotechnology Co., Ltd. (Dalian, China). FAP1 Ab-AF5344 and alpha-SMA Ab-AF1032 were obtained from Affinity Biosciences LTD (Jiangsu, China).
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