To identify the prophylactic effect of DHA on tBHP-induced autophagosomes and autolysosomes, the IFRS1 cells were plated onto 8-well chamber slides (Watson) (5 × 104 cells/well) and cultured in a humidified atmosphere containing 5% CO2 at 37 °C for 12 h prior to a treatment. The IFRS1 cells were pretreated with 10 μM DHA for 12 h and cultured with 300 μL of 25 nM DAPRed (Dojindo, Kumamoto, Japan), 200 nM DALGreen (Dojindo, Kumamoto, Japan), and 1 μg/mL Hoechst 33342 (Thermo Fisher Scientific, Waltham, MA, USA) working solutions for 30 min. After the cells reacted with the reagents, they were washed twice with DMEM/F12 containing 1% FBS and an N2 supplement medium. The cells were cultured with 50 μM tBHP for 3 h then washed twice with a Live Cell Imaging Solution (Thermo Fisher Scientific, Waltham, MA, USA) and observed with a fluorescence microscope (BZ-X810) (Keyence, Osaka, Japan). The fluorescence images were analyzed using BZ-X800 Analyzer software version 1.1.1.8 (Keyence, Osaka, Japan).
Dalgreen
Manufactured by Dojindo Laboratories
Sourced in Japan
DALGreen is a fluorescent dye for detecting active lysosomes in live cells. It is a cell-permeable dye that selectively accumulates in active lysosomes and exhibits green fluorescence upon excitation.
Automatically generated - may contain errors
Lab products found in correlation
Dapred, by Dojindo Laboratories (5 mentions)
Live cell imaging solution, by Thermo Fisher Scientific (3 mentions)
Cyto id, by Enzo Life Sciences (2 mentions)
Anti nlrp3 and anti cd80, by Santa Cruz Biotechnology (2 mentions)
Anti fade solution containig dapi, by Solarbio (2 mentions)
Goat serum, by Solarbio (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Lsm 780, by Zeiss (2 mentions)
Lsm 700 confocal microscope, by Zeiss (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Bz x800 analyzer, by Keyence (1 mentions)
Bz x810, by Keyence (1 mentions)
Rapamycin, by Cell Signaling Technology (1 mentions)
Dapgreen, by Dojindo Laboratories (1 mentions)
T8787, by Merck Group (1 mentions)
Hcs nuclearmask blue stain, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade mountant, by Thermo Fisher Scientific (1 mentions)
Eclipse ti2, by Zeiss (1 mentions)
Magic red, by ImmunoChemistry Technologies (1 mentions)
Bz x800, by Keyence (1 mentions)
14 protocols using dalgreen
1
DHA Protects Cells from Oxidative Stress
2
Quantification of Autolysosomes in Cells
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The autolysosome was detected using the method of fluorescence assay described as previously published [46 (link)]. Briefly speaking, the total number of 150,000 cells were plated in 24-well plate and co-cultured at 37 °C with 250 μL of 1 μM DALGreen (D675, Dojindo molecular technologies) for 30 min. Then, the cells were washed twice with DMEM/F12 growth medium and cultured in the medium containing different concentrations of magnesium. The activation of autophagy was induced with 10 μM Rapamycin (9904S, Cell Signaling Technology). Subsequently, intracellular autolysosomes were observed by fluorescence microscope, and the number of autolysosomes per cell was blinded-counted by two experienced collaborators.
3
DALGreen Staining Protocol
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DALGreen staining was performed based on previous publication with some modifications (Iwashita et al., 2018 ). A total of 200 000 cells were planted in 24‐well plate and cultured at 37 °C with 1 mL of 0.5 μm DALGreen (Dojindo Laboratories, Minato‐ku, Tokyo, Japan) working solution for 30 min. After the cells were washed twice with RPMI 1640 medium, they were observed under fluorescence microscope.
4
Autophagy Detection in Cancer Cells
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Cells were plated in confocal dishes and cultured at 37 °C with 1 mL of a 0.6 μM DALGreen [25 (link)] (Dojindo Laboratories, Shanghai, China) working solution for 30 min. After the cells were washed twice with culture medium, they were incubated with 50 μM CDDP, with or without 5 mM 4-PBA [26 (link)] or 5 mM 3-MA [27 (link)]. 8 h later, cells were washed twice with Hanks’ HEPES buffer and observed with a FluoView FV1000 confocal microscope. Cells with more than three DALGreen-positive foci were considered to be autophagy-positive cells.
5
Autophagy Dye-Based Quantification
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According to user instructions, autophagy-detecting dyes (Dojindo, Kumamoto, Japan), such as 12.5 nM DAPGreen [35 (link)] and 125 nM DALGreen [36 (link)], were chosen to probe autophagosomes/autolysosomes and autolysosomes, respectively. After PBS washing, drug-treated cells were detected and quantified by flow cytometry.
6
Visualizing Autophagy Dynamics in Cells
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For immunofluorescence and confocal microscopy, cells were fixed in 4% paraformaldehyde and permeabilized using 0.1% Triton X-100 (T8787, Sigma). After extensive washing, cells were analyzed under confocal microscopy (LSM780, Zeiss). Detection of autophagosomes and autolysosomes in IMR-90 cells was done by staining with DAPRed and DALGreen (Dojindo Molecular Technologies), following the manufacturer’s guidelines. DAPRed is a hydrophobic dye that fluoresces in hydrophobic conditions found in autolysosomes and autophagosomes, while DALGreen is a hydrophobic dye that emits fluorescence in the acidic conditions of autolysosomes. Staining solution containing DAPRed and DALGreen at final concentration of 0.5 μM DAPRed and 1 μM DALGreen was added to cell monolayers, followed by incubation at 37° C for 30 min. After incubation, cells were rinsed twice with the culture medium, prior to treatment with the medium, GL or sub-fractions. Stained cells were further incubated at 37° C for 16 hrs, prior to confocal microscopy observation.
7
Phagosome-Lysosome Fusion Detection
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DALGreen (Dojindo Molecular Technologies, Inc) was used for the detection of phagosome-lysosome fusion. In several experiments, DALGreen was co-stained with a lysosomal marker, LysoTracker Red. After DALGreen was stained, cells were washed with PBS three times and stained with Hoechst 33342 dye as an index of the nucleus. The resulting fluorescence was visualized by fluorescent microscopy (Evos, Hatfield, PA, USA).
8
Multimodal Analysis of Cell Death in Cardiomyocytes
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H9c2 cells and adult rat cardiomyocytes, which were isolated as described previously
27 (link) were seeded on glass coverslips. Staining using different dyes including Cyto‐ID, (1:1000, Enzo Life Sciences, #51031), Magic Red (1:260, ImmunoChemistry Technologies, #942), DalGreen (1:1000, Dojindo, #D675) DapRed (1:1000, Dojindo, #D677), cleaved caspase 3/7 (1:500, ThermoFisher, #C10423), and ReadyProbe (ThermoFisher, #R37609) was performed according to manufacturer's protocols and DAPI (HCS NuclearMask Blue Stain, ThermoFisher, #H10325) was added simultaneously. Cells were fixed in 10% formalin (Sigma, #HT501128) after treatment and mounted with ProLong Gold Antifade Mountant (ThermoFisher, #P36930). Slides were observed under Nikon Eclipse Ti2, Zeiss LSM700 confocal microscope at 60×, Evos FL Auto 2 and for continuous measurements Cellnsight CX7 Platform (ThermoFisher, #CX7A1110) was used. Co‐localization analysis was performed with an ImageJ JACOP plug‐in. At least five random fields from one coverslip/well, counting at least 30 cells were average in each independent experiment. The bar chart represents mean ± SEM values from at least three independent experiments.
27 (link) were seeded on glass coverslips. Staining using different dyes including Cyto‐ID, (1:1000, Enzo Life Sciences, #51031), Magic Red (1:260, ImmunoChemistry Technologies, #942), DalGreen (1:1000, Dojindo, #D675) DapRed (1:1000, Dojindo, #D677), cleaved caspase 3/7 (1:500, ThermoFisher, #C10423), and ReadyProbe (ThermoFisher, #R37609) was performed according to manufacturer's protocols and DAPI (HCS NuclearMask Blue Stain, ThermoFisher, #H10325) was added simultaneously. Cells were fixed in 10% formalin (Sigma, #HT501128) after treatment and mounted with ProLong Gold Antifade Mountant (ThermoFisher, #P36930). Slides were observed under Nikon Eclipse Ti2, Zeiss LSM700 confocal microscope at 60×, Evos FL Auto 2 and for continuous measurements Cellnsight CX7 Platform (ThermoFisher, #CX7A1110) was used. Co‐localization analysis was performed with an ImageJ JACOP plug‐in. At least five random fields from one coverslip/well, counting at least 30 cells were average in each independent experiment. The bar chart represents mean ± SEM values from at least three independent experiments.
9
Dual Fluorescence Autophagy Monitoring
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DALGreen and DAPRed (Dojindo) were used to stain autophagosomes with enhanced fluorescence in hydrophobic environments. DALGreen fluorescence is enhanced at an acidic pH and is suitable for monitoring the autophagy degradation stage (autolysosomes). In contrast, DAPRed has a pH-independent fluorescence profile and remains fluorescent at an almost constant intensity throughout autophagy (autophagosome). Cells seeded in glass bottom dishes were stained with DALGreen and DAPRed for 30 min at 37°C. The medium was replaced with HBSS and the intensities of the green and red signals were measured and calculated (BZ-X800, Keyence).
10
Fluorescence Imaging of Cell Viability
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DALGreen and DAPRed (Dojindo Molecular Technologies, Kumamoto, Japan) were used according to the supplier's protocol as described below. The CRL-1623 cells were preincubated in a culture medium containing 1 μM DALGreen and 0.1 μM DAPRed for 30 min. After washing out the staining medium, the cells were treated with various concentrations of MT (0, 0.5, 1, and 2.5 mmol/L) for 48 h. Subsequently, fluorescence images were captured using a fluorescence microscope under identical conditions (same laser power and detector gains). The method was adapted from previously published research and has been appropriately cited in our study [30 ].
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