Acridine orange staining was performed to detect autophagy according to published protocol.36 , 37 Briefly, cells were treated with DMSO or PR‐619 (8 µmol/L) for 24 hours and stained with Acridine orange (Solarbio, China) in PBS containing 5% FBS at 37°C for 30 minutes. Cells were washed and observed under fluorescence microscopy (magnification: 200×; Nikon, Nikon Inc, Tokyo, Japan). The formation of acidic vesicular organelles (AVOs) was examined under fluorescence microscopy. AVOs, such as autolysosomes, were orange/red. Non‐AVO areas (cytoplasm, nucleus, and nucleolus) were green.
Acridine orange
Manufactured by Solarbio
Sourced in China
Acridine orange is a fluorescent dye commonly used in biological research. It can intercalate with DNA and RNA, emitting green or orange fluorescence depending on the nucleic acid structure. The dye is often used for staining and visualizing nucleic acids in various applications such as cell biology, molecular biology, and flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Cell counting kit 8 cck 8, by Dojindo Laboratories (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Monodansylcadaverine mdc, by Solarbio (2 mentions)
Chloroquine cq, by Merck Group (1 mentions)
Anti lc3a b, by Cell Signaling Technology (1 mentions)
Rapamycin rapa, by MedChemExpress (1 mentions)
Alexa fluor 488 conjugated goat anti rabbit igg, by Beyotime (1 mentions)
Anti rabbit igg sa00001 2, by Proteintech (1 mentions)
N n methylenebis acrylamide mba, by Merck Group (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Glycerol, by Maclin Biochemical Technology (1 mentions)
4t1 mouse breast cancer cells, by American Type Culture Collection (1 mentions)
N isopropylacrylamide nipam, by Maclin Biochemical Technology (1 mentions)
Malondialdehyde mda content assay kit, by Solarbio (1 mentions)
Catalase cat activity assay kit, by Solarbio (1 mentions)
2 7 dichlorodihydrofluorescein diacetate dcfh da, by Solarbio (1 mentions)
Fitc annexin 5 apoptosis detection kit 1, by BD (1 mentions)
Phospho akt, by Cell Signaling Technology (1 mentions)
Sqstm1 p62, by Cell Signaling Technology (1 mentions)
27 protocols using acridine orange
1
Acridine Orange Staining for Autophagy
2
Chloroquine and Rapamycin Autophagy Modulation
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Chloroquine (CQ) was purchased from Sigma-Aldrich (St. Louis, MI, USA). Rapamycin (Rapa) was purchased from MedChemExpress, (South Brunswick, NJ, USA). Acridine orange (AO) was purchased from Solarbio (Beijing, China). EG was purchased from Macklin (Shanghai, China).
The antibodies used in this study were anti-Fragilis (Huabio, Hangzhou, China), anti-LC3A/B (Cell Signaling Technology, Danvers, MA, USA), anti-cathepsin B polyclonal antibody (Wanleibio, Shenyang, China), anti-BVDV E2-specific mouse monoclonal antibody (VMRD, Pullman, WA, USA). Additionally, anti-LAMP1 (21997-1-AP), anti-SQSTM1/p62 (18420-1-AP), anti-GAPDH (60004-1-AP), and anti-Tubulin (10068-1-AP) were purchased from ProteinTech Group Inc. (Rosemont, IL, USA). Secondary antibodies in this study included Alexa Fluor 488-Conjugated Goat Anti-Rabbit IgG (Beyotime, Shanghai, China). Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (SA00001-1), and anti-rabbit IgG (SA00001-2) were purchased from ProteinTech Group Inc. (Rosemont, IL, USA).
The antibodies used in this study were anti-Fragilis (Huabio, Hangzhou, China), anti-LC3A/B (Cell Signaling Technology, Danvers, MA, USA), anti-cathepsin B polyclonal antibody (Wanleibio, Shenyang, China), anti-BVDV E2-specific mouse monoclonal antibody (VMRD, Pullman, WA, USA). Additionally, anti-LAMP1 (21997-1-AP), anti-SQSTM1/p62 (18420-1-AP), anti-GAPDH (60004-1-AP), and anti-Tubulin (10068-1-AP) were purchased from ProteinTech Group Inc. (Rosemont, IL, USA). Secondary antibodies in this study included Alexa Fluor 488-Conjugated Goat Anti-Rabbit IgG (Beyotime, Shanghai, China). Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (SA00001-1), and anti-rabbit IgG (SA00001-2) were purchased from ProteinTech Group Inc. (Rosemont, IL, USA).
3
Synthesis and Characterization of Stimuli-Responsive Nanoparticles
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A gallium (Ga) and indium (In) were purchased from local company (China). N-Isopropylacrylamide (NIPAm) and glycerol were obtained from Shanghai Macklin Biochemical Co. Ltd. (Shanghai, China). N,N′-Methylenebis(acrylamide) (MBA) was purchased from Sigma-Aldrich (WUXI) Life Science & Technology Co. Ltd. (Wuxi, China). Ammonium persulfate (APS) was obtained from Aladdin Industrial Corporation (Shanghai, China). Doxorubicin hydrochloride (DOX·HCl) was purchased from Shanghai D&B Biological Science and Technology Co. Ltd. (Shanghai, China). All the chemicals were pure and used directly without further purifying. Dulbecco's Modification of Eagle's Medium (DMEM) was purchased from Wisent (China). Fetal bovine serum (FBS), penicillin/streptomycin and phosphate buffered saline (PBS, pH 7.4) were obtained from Thermo Fisher Scientific Corporation (China). Cell counting kit-8 (CCK-8) was purchased from Dojindo Laboratories (China). Acridine orange (AO) and ethidium bromide (EB) solution were obtained from Solarbio (China). 4T1 mouse breast cancer cells were supplied by American Type Culture Collection (ATCC).
4
Antioxidant and Hypoglycemic Effects of Panax quinquefolius
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The herb was purchased from the herb market (Guo Xin Pharmacy, Jiangsu, China) and identified as the root of Panax quinquefolius L. by Prof. Cunli Zhang of Northwest Agriculture and Forestry University of Science and Technology. We purchased 2-Deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl) amino]-D-glucose (2-NBDG) from Shanghai Dibai Biotechnology Co., Ltd. (Shanghai, China). N-Phenylthiourea (PTU), tricaine, alloxan, 2,7-Dichlorodihydrofluorescein diacetate (DCFH-DA), acridine orange (AO), riboflavin, catalase (CAT) activity assay kit and malondialdehyde (MDA) content assay kit were purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China).
5
Nitidine Chloride Induces Autophagy
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Nitidine chloride (NC, CAS no. 13063-04-2) was ordered from Tauto Biotech Co., Ltd. (Shanghai, China). 3-[4 (link),5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay was purchased from Beyotime Institute of Biotechnology (Shanghai, China), FITC Annexin V Apoptosis Detection Kit I(#556547) was purchased from BD Biosciences (Franklin Lakes, NJ, USA). Chloroquine (CQ) was ordered from Selleck. Monodansylcadaverine (MDC) or acridine orange (AO) were purchased from Solarbio Science & Technology Co., Ltd. (Beijing, China). The primary antibodies were provided by Cell Signaling Technology (USA), including LC3A/B (D3U4C, #12741, 1:1000), SQSTM1/p62 (D5E2, #8025, 1:1000), Akt (C67E7, #4691, 1:1000), Phospho-Akt (Thr308, D25E6, #13038, 1:1000), mTOR (7C10, #2983, 1:1000), Phospho-mTOR (Ser2448, D9C2, #5536, 1:1000), p70 S6 Kinase (49D7, #2708, 1:1000), Phospho-p70 S6 Kinase (Thr389, 108D2, #9234, 1:1000), 4E-BP1 (53H11, #9644, 1:1000), Phospho-4E-BP1 (Thr37/46, 236B4, #2855, 1:1000), β-Actin (13E5, #4970, 1:5000).
6
Acridine Orange Staining of Acidic Vesicles
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Acridine Orange (AO) (Solarbio, Beijing, China) is an important weak alkaline stain for detecting the structure of acidic vesicles. Cells in 6-well culture dishes were incubated with ethionine and MnTMPyP. Then, cells were washed twice with PBS, and then resuspended in 0.5 ml DMEM and 5% FBS with acridine orange dye working solution for 15 min at 37°C. The cells were visualized using fluorescence microscope (Nikon, Tokyo, Japan) and the data were analyzed with ImageJ Software.
7
Acridine Orange Staining of 786-O Cells
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The 786-O cells were seeded (1∗104) in 24-well plates for adherence and treated with different groups of drugs for 24 hours. Before staining, each well was washed with PBS for 15 minutes, three times, and the cells were fixed using 4% poly-formalin for 10 minutes. The dye acridine orange (AO) (Solarbio, China, diluted to 10 μg/mL before staining) was added to each well. After 15 minutes, each well was again washed with PBS for 15 minutes, three times. All the wells were observed using a fluorescent optical microscope in the mode of fluorescence. The nuclei were dyed by AO in green, and the fluorescence of the lysosomes was in orange [25 (link)].
8
Autophagy Regulation by Acridine Orange and N-Acetyl-L-Cysteine
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Acridine orange (AO) was from Solarbio. N-Acetyl-L-cysteine (NAC) was from Sigma. Bicinchoninic acid (BCA) protein assay kit and enhanced chemiluminescence kit were from Cwbio. RIPA buffer and LysoTracker Deep Red were from Beyotime Biotechnology. For these compounds, catalog information is listed in Table 1 . Western blotting and immunofluorescence staining used the following primary antibodies: anti-CTSL/major excreted protein and anti-CTSD from ABclonal Technology; anti-light chain 3 (LC3) B from Beyotime (Shanghai, China); anti-Sequestosome 1/p62 (SQSTM1/p62), anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH), anti-lysosome-associated membrane protein 2 (LAMP2), anti-β-actin, anti-α-tubulin, and anti-Beclin 1, from Protein tech; and Peroxidase-Conjugated AffiniPure Goat Anti-mouse IgG and goat anti-rabbit IgG from Cwbio. Catalog and dilution information of these reagents are listed in Table 2 .
9
Hydrothermal Synthesis of Functionalized HNTs
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HNTs were provided by Guangzhou Runwo Materials Technology Co., Ltd., China and used without further purification. The elemental composition of used HNTs by X-ray fluorescence (XRF) was determined as follows (wt%): SiO2, 58.91; Al2O3, 40.41; Fe2O3, 0.275; P2O5, 0.138; TiO2, 0.071. The weight loss of HNTs was 19.20%, which was mainly occurred in the temperature range of 400–500 °C. Fluorescein isothiocyanate (FITC) (analytical grade) was purchased from Shanghai Yeasen Biotechnology Co., Ltd., Shanghai, China. Indocyanine green (ICG) and streptavidin (SA) were purchased from Sigma-Aldrich (St. Louis, MO, US). Anti-EpCAM aptamer was purchased from Sangon Biotech, Shanghai. Acridine orange (AO) and ethidium bromide (EB) were purchased from Solarbio Science and Technology Co., Ltd., Beijing, China. Ultrapure water was obtained from Milli-Q water system. All the other chemicals were analytically graded and used directly without further purification.
10
Evaluation of Cellular Responses in HK-2 Cells
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Reagents:
DMEM/F-12 culture medium and fetal bovine serum were purchased from
Gibco. The CCK-8 kit was purchased from Dojindo Laboratory (Kumamoto,
Japan). Cell membrane red fluorescent probe 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine
perchlorate (DiI), 4,6-diamidino-2-phenylindole (DAPI) staining solution,
and Fluo-4 AM were purchased from Shanghai Biyuntian Biotechnology
Co., Ltd. Annexin V-FITC/propidium iodide (PI), 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimi-dazolylcarbocyanine
iodide (JC-1), and 2′,7-dichlorodihydrofluorescein diacetate
(DCFH-DA) were purchased from KeyGEN BioTECH Co. Ltd. (Nanjing, China).
Acridine orange (AO) and monodansylcadaverine (MDC) were purchased
from Beijing Solarbio Technology Co., Ltd. Human kidney proximal tubular
epithelial (HK-2) cells were purchased from the Shanghai Cell Bank
of the Chinese Academy of Sciences (Shanghai, China).
Instruments:
multifunctional microplate reader (SafireZ, Tecan, Switzerland); flow
cytometer (FACS Aria, BD, USA); inverted fluorescence microscope (OLYMPUS,
U-HGLGPS, Japan); laser confocal microscope (LSM510 META DUO SCAN,
ZEISS, Germany); and optical microscope (OLYMPUS, TH4-200, Japan).
DMEM/F-12 culture medium and fetal bovine serum were purchased from
Gibco. The CCK-8 kit was purchased from Dojindo Laboratory (Kumamoto,
Japan). Cell membrane red fluorescent probe 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine
perchlorate (DiI), 4,6-diamidino-2-phenylindole (DAPI) staining solution,
and Fluo-4 AM were purchased from Shanghai Biyuntian Biotechnology
Co., Ltd. Annexin V-FITC/propidium iodide (PI), 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimi-dazolylcarbocyanine
iodide (JC-1), and 2′,7-dichlorodihydrofluorescein diacetate
(DCFH-DA) were purchased from KeyGEN BioTECH Co. Ltd. (Nanjing, China).
Acridine orange (AO) and monodansylcadaverine (MDC) were purchased
from Beijing Solarbio Technology Co., Ltd. Human kidney proximal tubular
epithelial (HK-2) cells were purchased from the Shanghai Cell Bank
of the Chinese Academy of Sciences (Shanghai, China).
Instruments:
multifunctional microplate reader (SafireZ, Tecan, Switzerland); flow
cytometer (FACS Aria, BD, USA); inverted fluorescence microscope (OLYMPUS,
U-HGLGPS, Japan); laser confocal microscope (LSM510 META DUO SCAN,
ZEISS, Germany); and optical microscope (OLYMPUS, TH4-200, Japan).
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