To assess the intracellular level of glutathione (GSH), ThiolTracker (T10095, Thermo Fisher Scientific, Waltham, MA, USA) was used as per the manufacturer’s protocol. Analysis was performed using a FACS Canto II flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA).
Thioltracker
Manufactured by Thermo Fisher Scientific
Sourced in United States
ThiolTracker is a fluorescent probe designed to detect and quantify thiol-containing molecules in biological samples. It functions by selectively reacting with thiol groups, resulting in a fluorescent signal that can be measured using appropriate instrumentation.
Automatically generated - may contain errors
Lab products found in correlation
Mitotracker, by Thermo Fisher Scientific (2 mentions)
Bodipy fl maleimide, by Thermo Fisher Scientific (2 mentions)
Alexa 555 plus, by Thermo Fisher Scientific (2 mentions)
Prolong gold, by Thermo Fisher Scientific (2 mentions)
Lsm710 elyra p1, by Zeiss (2 mentions)
Facscanto 2 flow cytometer, by BD (1 mentions)
Mitosox, by Thermo Fisher Scientific (1 mentions)
Image it lipid peroxidation kit, by Thermo Fisher Scientific (1 mentions)
Cellrox, by Thermo Fisher Scientific (1 mentions)
Hrp conjugated streptavidin, by Abcam (1 mentions)
Rabbit anti calnexin, by Merck Group (1 mentions)
Ros glo assay, by Promega (1 mentions)
Glutathione (gsh), by Beyotime (1 mentions)
Albumin fluorescein isothiocyanate conjugate fitc bsa, by Merck Group (1 mentions)
Brusatol, by MedChemExpress (1 mentions)
Ros detection kit, by Abcam (1 mentions)
Lipopolysaccharide lps, by Merck Group (1 mentions)
Atp detection kit, by Beyotime (1 mentions)
Alpha plan apo 100 1.46 oil objective lens, by Zeiss (1 mentions)
Plan apochrombat 63x 1, by Zeiss (1 mentions)
6 protocols using thioltracker
1
Intracellular Glutathione Quantification
2
Multiparametric Flow Cytometry Analysis
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A2780CIS or PEO4 cells were cultured in either unstimulated conditions or treated with monotherapies or combinations of saline (vehicle), 16 µM cisplatin (CIS), or 1.5 µM ivermectin (IVM) for 36 h. Live A2780CIS or PEO4 cells were collected and stained with Tetramethyl rhodamine, ethyl ester (TMRE; Abcam; #AB113852, Cambridge, UK), MitoTracker (ThermoFisher # M7514, Waltham, MA, USA), MitoSOX (ThermoFisher # M36008, Waltham, MA, USA), CellROX (ThermoFisher #C10444, Waltham, MA, USA), ThiolTracker (ThermoFisher # T10095, Waltham, MA, USA), or Image—iT Lipid Peroxidation kit (ThermoFisher #C10445, Waltham, MA, USA) in flow buffer (PBS supplemented with 1% BSA and 2 mM EDTA) for 10–30 min at room temperature, according to the manufacturers’ recommendations. For the Image—iT Lipid Peroxidation kit (ThermoFisher #C10445, Waltham, MA, USA), lipid peroxidation was measured through ratio analysis of 510 nM to 590 nM emission fluorescence from the kit’s dye, as per manufacturer’s instructions. Cells were then washed with flow buffer followed by acquisition with the ZE5 flow cytometer cell analyzer (Bio-Rad, Hercules, CA, USA) or the FACSymphony flow cytometer cell analyzer (BD Biosciences, San Jose, CA, USA). 500,000 events were analyzed per group using FlowJo software (version 10.4.2; BD Biosciences, San Jose, CA, USA).
3
Measurement of Intracellular ROS in Cells
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Intestinal epithelial cells SKCO15 were a kind gift from Dr. Charles Parkos, and were maintained in high glucose DMEM containing 10% fetal bovine serum, additionally supplemented with non-essential amino acids and L -glutamine. The mouse Rac-1 antibody was purchased from BD Transduction. The HydroCy3 was obtained from LI-Core (sold as ROSstar 550), and the Thiol Tracker from Thermo Scientific and were both used as directed in the manufacturers’ instructions and images acquired using a standard fluorescence microscope with camera. The biotinylated iodoacetamide was obtained from Anaspec. Anti-mouse secondary antibody conjugated to HRP was obtained from GE, while the streptavidin conjugated HRP was obtained from Abcam. Rabbit anti-calnexin was purchased from Sigma-Aldrich. ROS-GLO assay was purchased from Promega and performed in a 96-well format following the manufacturer’s instructions on overnight cultures of bacterial supernatants.
4
Dexmedetomidine Modulates Oxidative Stress
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Dexmedetomidine was purchased from Hengrui (Jiangsu, China). Albumin-fluorescein isothiocyanate conjugate (FITC-BSA) and Lipopolysaccharide (LPS) were purchased from Sigma (St. Louis, MO, US). Antibodies for G6PD, GPX4, ZO-1, β-actin, and ROS detection kit were purchased from Abcam (Cambridge, MA, America). Antibody for Nrf2 was purchased from GeneTEX (San Antonio, TX, America). Lipid Peroxidation kit, MITO-SOX detection kit, Mito-tracker and Thiol-tracker were purchased from Thermo Scientific (Waltham, MA, America). Glutathione, MDA, and ATP detection kit were purchased from Beyotime Biotechnology (Shanghai, China). Brusatol and Erastin were purchased from MedChemExpress (Monmouth, NJ, America). All other chemicals were purchased from Sigma unless specifically mentioned otherwise.
5
Sperm Immunofluorescence Labeling Protocol
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As described previously (59 (link), 66 (link)), mouse sperm were washed in PBS twice, attached on the glass coverslips, and fixed with 4% paraformaldehyde (PFA) in PBS at room temperature (RT) for 10 min. Fixed samples were permeabilized using 0.1% Triton X-100 in PBS at RT for 10 min, washed in PBS, and blocked with 10% goat serum in PBS at RT for 1 h. Cells were stained with antibody in PBS supplemented with 10% goat serum at 4 °C overnight. After washing in PBS, the samples were incubated with goat anti-rabbit Alexa 647 or Alexa 555-plus (Invitrogen, 1:1000) in 10% goat serum in PBS at RT for 1 h. Hoechst dye was used to stain nucleus to indicate sperm head. For BODIPY-N-ethylmaleimide labeling, reduced thiols within proteins were alkylated with BODIPY FL maleimide (final concentration of 10 nM, ThermoFisher) for 30 min in the dark after permeabilization. The sample was then quenched by the addition of 500 mM 2-mercaptoethanol for 30 min in the dark, followed by three times washing in PBS. For ThiolTracker (ThermoFisher) labeling, fixed sperm were stained with 20 μM dye, followed by three times washing in PBS. Stained sperm samples were mounted with Prolong gold (Invitrogen) and cured for 24 h, followed by imaging with Zeiss LSM710 Elyra P1 using Plan-Apochrombat 63×/1.40 or alpha Plan-APO 100×/1.46 oil objective lens (Carl Zeiss).
6
Immunolabeling of Mouse and Human Sperm
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Mouse and human sperm were washed in PBS twice, attached on the glass coverslips, and fixed with 4% paraformaldehyde (PFA) in PBS at room temperature (RT) for 10 min (mouse) or at 4°C for 1 hr (human). Fixed samples were permeabilized using 0.1% Triton X-100 in PBS at RT for 10 min, washed in PBS, and blocked with 10% goat serum in PBS at RT for 1 hr. Cells were stained with antibody in PBS supplemented with 10% goat serum at 4°C overnight. After washing in PBS, the samples were incubated with goat anti-rabbit Alexa 647 or Alexa 555-plus (Invitrogen, 1:1,000) in 10% goat serum in PBS at RT for 1 hr. Hoechst was used to counterstain nucleus for sperm head. For BODIPY-N-ethylmaleimide labeling, reduced thiols within proteins were alkylated with BODIPY FL maleimide (final concentration of 10 nM, ThermoFisher) for 30 min in the dark after permeabilization. The sample was then quenched by the addition of 500 mM 2mercaptoethanol for 30 min in the dark, followed by 3 times washing in PBS. For ThiolTracker (ThermoFisher) labeling, fixed sperm were stained with 20 μM dye, followed by 3 times washing in PBS. Stained sperm samples were mounted with Prolong gold (Invitrogen) and cured for 24 hr, followed by imaging with Zeiss LSM710 Elyra P1 using Plan-Apochrombat 63X/1.40 or alpha Plan-APO 100X/1.46 oil objective lens (Carl Zeiss).
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