Adult fat bodies (2–3 days old) were dissected in 1X PBS and stained with either LysoTracker Red (1:1000, ThermoFisher L7528) or Magic Red (1:150, BioRad IT937) Mitotracker (1:300, ThermoFisher M7512) for 5, 15 and 30 minutes respectively. Samples were directly visualized using Zeiss Axio Imager fluorescent microscope.
Magic red
Manufactured by Bio-Rad
Sourced in United States
Magic Red is a fluorescent caspase detection reagent manufactured by Bio-Rad. It is used for the detection of caspase activity in live cells.
Automatically generated - may contain errors
Lab products found in correlation
Axio imager fluorescent microscope, by Zeiss (1 mentions)
Lysotracker red, by Thermo Fisher Scientific (1 mentions)
Mitosox red mitochondrial superoxide indicator, by Thermo Fisher Scientific (1 mentions)
Bodipy 493 503, by Thermo Fisher Scientific (1 mentions)
Mitotracker red cmxros, by Thermo Fisher Scientific (1 mentions)
Acridine orange, by Bio-Rad (1 mentions)
Ab4074, by Abcam (1 mentions)
Alexa fluor conjugates, by Thermo Fisher Scientific (1 mentions)
Vectashield reagent, by Vector Laboratories (1 mentions)
5 protocols using magic red
1
Fluorescent Staining of Adult Fly Fat Bodies
2
Imaging Live Cells with Magic Red
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Cells were plated in 24-mm round coated glass coverlips (Marienfeld, 0101060) the day before the experiment. The incubation of the cells with Magic Red (Bio-Rad, ICT937) took 20 min, after which the coverslip was washed in warm imaging buffer and then placed on a coverslip holder filled with pre-heated imaging buffer. The coverslip was then immediately taken to the Nikon/PerkinElmer Spinning Disk microscope under a humidified chamber heated at 30°C, and imaged. Data was analyzed using ImageJ.
3
Multimodal Lysosomal Imaging Approach
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BODIPY™ 493/503, LipidTOX™, and MitoTracker Red CMX ROS, MitoSOX™ Red Mitochondrial Superoxide Indicator (Thermo Fisher Scientific) (Thermo Fisher Scientific) were used according to the manufacturer’s guidelines. Cathepsin‐reactive lysosomes were visualised using Magic Red (Bio‐Rad, ICT937) according to the manufacturer’s guidelines and as described (Bright et al,2016 (link)) with minor modifications. Briefly, cells were seeded 48 h before the measurement and treated with DFP ± DGAT1/2 inhibitors as previously outlined above before co‐incubation with Magic Red™ (1:26 dilution of stock solution as per manufacturer's protocol) for 30 and 5 min with Hoescht 33342 (1 µg/ml) followed by live‐cell imaging as described below.
4
Autophagy Evaluation in Nanomaterial-Treated Cells
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Cells were seeded with a density of 62 × 103 cm2, and after reaching confluence, they were stimulated with Ag- and Au-NPs ± PT for 24 h, BafA 50 nM for 2 h or left with medium alone. Acridine orange (Bio-Rad, Hercules, CA, USA) at the concentration of 1 µg/mL was then added to the cells and incubated for 30 min. Cells were washed with PBS, and fluorescence signals (both red and green) were detected with a plate reader (excitation 480 nm; emission 620 nm for red, 550 nm for green). Data are presented as red/green signals ratio as previously described [38 (link),39 (link)]. For the Magic Red assay, HIEC-6 cells were exposed to treatments for 24 h, leupeptin 20 nM for 2 h or left with medium alone. Magic Red (Bio-Rad, Hercules, CA, USA) was then added to the cells and incubated for 30 min. Cells were washed with PBS, and fluorescence signals were detected with a plate reader (excitation 595 nm; emission 635). The unlabeled cells and the noncellular test, run as negative controls, showed null or negligible signals.
5
Immunofluorescence Staining of Endosomal Markers
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Mouse monoclonal antibodies against CD63 (H5C6; BD; clone TS63b, gift of Eric Rubinstein, Centre d'Immunologie et des Maladies Infectieuses, Paris, France) were used at 1:200 and 1:2,000, respectively, and mouse monoclonal against CD9 (clone TS9 36; gift of Eric Rubinstein) was used 1:2,000. Polyclonal antibody against LAMP1 (PA1-654A; Thermo Fisher Scientific) was used 1:100. Rabbit monoclonal antibody against ORP1L (ab132265; Abcam) was used 1:1,000. Rabbit polyclonal antibody against alpha-Tubulin (ab4074; Abcam) was used 1:4,000. MagicRed (ICT 938; Bio-Rad) was reconstituted according to the manufacturer’s recommendation and added to the cells at a final dilution of 1:2,600 5 min before live-cell imaging. For immune-fluorescence, cells were seeded on coverslips, fixed in 2% PFA (in PBS) for 20 min at RT, incubated with blocking/washing buffer (1X PBS/1% BSA/0.1% Saponin) for 1 h at RT. The slides were incubated with primary antibody for 45 min, secondary antibody (AlexaFluor conjugates, Thermo Fisher Scientific) at 1:2,500 for 45 min at RT, mounted with the Vectashield reagent (Vector Laboratories), and sealed with nail polish.
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