Mitochondrial O2.−, total H2O2 and free cytosolic Ca2+ levels were determined as reported before23 (link), using MitoSOX fluorescent probes (#M36008; Thermo Fisher), H2DCF-DA (#C6827; Thermo Fisher) and Fluo-4 (#F23917; Thermo Fisher). Briefly, HaCaT cells were seeded in 96 well plates and incubated up to 24 h or 7 d with sFLG in the range 0.5 to 100 µg/mL. After treatment, cells were washed twice with PBS then loaded for 30 min with the fluorescent probe (one independent probe per assay; 1 µM MitoSOX and Fluo-4; 2.5 µM H2DCFDA) and imaged with a Nikon TiU microscope (20 × objective). Pictures were analyzed and processed with ImageJ 1.53. The results show the percentage of cell signal vs. control (n = 4). Tracking experiments were performed by monitoring fluorescence levels at different times in the same cells using a Zeiss LSM-600 confocal microscope (63 × objective). Pictures were processed with Zen 2012 blue edition and ImageJ 1.53. Results show the variation of fluorescence from time 0 (> 25 cells/experiment).
Mitosox fluorescent probe
Manufactured by Thermo Fisher Scientific
Sourced in United States
MitoSOX fluorescent probes are a family of mitochondrial-targeted superoxide indicators. They are designed to detect superoxide in the mitochondria of live cells.
Automatically generated - may contain errors
Lab products found in correlation
H2dcfda, by Thermo Fisher Scientific (2 mentions)
Fluo 4, by Thermo Fisher Scientific (1 mentions)
Fluo 4, by Nikon (1 mentions)
Lsm 600 confocal microscope, by Zeiss (1 mentions)
Ti u microscope, by Nikon (1 mentions)
H2dcfda, by Nikon (1 mentions)
Seahorse xf assay media, by Agilent Technologies (1 mentions)
Seahorse xf media, by Agilent Technologies (1 mentions)
Viacount, by Merck Group (1 mentions)
Penicillin streptomycin, by Erba Group (1 mentions)
Bradford and rc dc protein assay kit, by Bio-Rad (1 mentions)
Seahorse xf96 microplate plates, by Agilent Technologies (1 mentions)
Mito stress kits, by Agilent Technologies (1 mentions)
N acetyl cysteine (nac), by Merck Group (1 mentions)
Metformin, by Merck Group (1 mentions)
H9c2 cardiomyoblasts crl 1446, by American Type Culture Collection (1 mentions)
Mitotracker green fm, by Thermo Fisher Scientific (1 mentions)
Eclipse e800, by Nikon (1 mentions)
Eclipse ts2, by Nikon (1 mentions)
Lsrfortessa x 20, by BD (1 mentions)
4 protocols using mitosox fluorescent probe
1
Mitochondrial ROS and Calcium Sensing
2
Cardiomyoblast Metabolic Profiling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Embryonic ventricular rat heart derived H9c2 cardiomyoblasts (CRL-1446) were from American Type Culture Collection (Manassas, USA); Penicillin-Streptomycin, Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), and Phosphate Buffered Saline (PBS) were from Carlo Erba (Milano, Italy); dichlorfluorescein diacetate (DCFH-DA) and MitoSOX fluorescent probes were from ThermoFisher Scientific (Waltham, USA); Bradford and RC DC protein assay kit was from Bio-Rad Laboratories (Hercules, USA); ViaCount was from Merck-Millipore (Darmstadt, Germany). Seahorse XF96 microplate plates, Seahorse XF Assay media, Seahorse XF media and Mito Stress Kits were from Agilent (Santa Clare, USA). All other chemicals including NAC and metformin were obtained from Sigma-Aldrich (St. Louis, USA).
3
Multimodal Mitochondrial Analysis in Endothelial Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
EC Immunofluorescence—MHECs were allowed to grow until 80% confluency and then processed for immunofluorescence of MnSOD, following the manufacturer’s protocol (Cell signaling Technology, USA). Slides were imaged at 20× objective on an upright microscope (Eclipse E800, Nikon Instruments Inc., USA) using spot Advanced™ Software (SPOT Imaging, USA).
Live-cell mitochondria and mitochondrial ROS fluorescence—MHEC were cultured to 80% confluency. Cells were thereafter incubated with 100 nM MitoTracker® Green FM to label mitochondria, and 5 µM MitoSOX™ fluorescent probe to estimate mitochondrial ROS production, following the manufacturer’s protocol (Thermo Fisher Scientific, USA) [50 (link)]. Each well was imaged at 20× objective on an inverted microscope (Eclipse Ts2, Nikon Instruments Inc., USA). Mitochondrial ROS fluorescence was also measured on a 96-well plate reader after labeling with MitoSOX.
Mito-roGFP assay—Mito-roGFP assay was performed as previously described [56 (link)]. Imaging was performed using a Nikon ECLIPSE TE20000-U microscope (Nikon Instruments Inc., NY, USA) with excitation and emission filters at 405/488 nm. The oxidative status was then quantified considering the maximal reduction and oxidation.
Live-cell mitochondria and mitochondrial ROS fluorescence—MHEC were cultured to 80% confluency. Cells were thereafter incubated with 100 nM MitoTracker® Green FM to label mitochondria, and 5 µM MitoSOX™ fluorescent probe to estimate mitochondrial ROS production, following the manufacturer’s protocol (Thermo Fisher Scientific, USA) [50 (link)]. Each well was imaged at 20× objective on an inverted microscope (Eclipse Ts2, Nikon Instruments Inc., USA). Mitochondrial ROS fluorescence was also measured on a 96-well plate reader after labeling with MitoSOX.
Mito-roGFP assay—Mito-roGFP assay was performed as previously described [56 (link)]. Imaging was performed using a Nikon ECLIPSE TE20000-U microscope (Nikon Instruments Inc., NY, USA) with excitation and emission filters at 405/488 nm. The oxidative status was then quantified considering the maximal reduction and oxidation.
4
Mitochondrial Superoxide Evaluation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The MitoSOX fluorescent probe (Thermo Fisher Scientific) was used to assess mitochondrial superoxide. Cells were cotreated with or without NTPAM and N-acetyl cysteine (NAC) (2 and 5 mM, respectively), then incubated at 37°C for 24 h. After treatment, cells were stained with MitoSOX (5 μM) in Hank's balanced salt solution for 15 min. Fluorescence-stained cells (2 × 105) were analyzed via flow cytometry (LSRFortessa X-20; BD Biosciences), and FlowJo software version 10 (Tree Star) was used to analyze the proportions of cells.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!