Aliquots (200 μl) of whole hemolymph (each containing about 1–2 × 106 cells/ml) were incubated with the OlyA6/PlyB mixture (25 and 50 μg/ml) for 30 min at 16°C. Control samples were run in parallel. Total hemocyte count was carried out using an Omnicyt flow cytometer (Cytognos SL, Salamanca, Spain). Samples were pelleted by centrifugation (100 × g for 10 min) and stained with different fluorophores. Annexin V-FITC (ANX)/propidium iodide (PI) and TMRE staining was carried out as previously described (Ciacci et al., 2012 (link); Canesi et al., 2015 (link)); MitoSOX and CM-H2DCFDA staining was performed as described above for confocal scanning laser microscopy. Samples were also analysed by the Intracellular Glutathione Detection Assay Kit (Abcam) (at ex: 490, em: 520 nm). Sample acquisition and analyses were performed by means of a FACS Canto II flow cytometer and analysed with DiVa™ software collecting at least 10,000 events for each sample. Data, representing the mean ± standard deviation (SD) of at least three experiments, are expressed as Mean Fluorescence Intensities reported as percent changes with respect to controls, except for ANX/PI staining, where data are expressed as percent positive cells with respect to controls.
Intracellular glutathione detection assay kit
Manufactured by Abcam
The Intracellular Glutathione Detection Assay Kit is designed to quantify the level of glutathione, a crucial antioxidant, within cells. This kit utilizes a fluorometric method to enable the measurement of glutathione concentration in a simple, sensitive, and reliable manner.
Automatically generated - may contain errors
Lab products found in correlation
2 7 dichlorodihydrofluorescein diacetate h2dcfda, by Thermo Fisher Scientific (3 mentions)
Mitosox, by Thermo Fisher Scientific (3 mentions)
Facscanto 2, by BD (3 mentions)
2 n 7 nitrobenz 2 oxa 1 3 diaxol 4 yl amino 2 deoxyglucose 2 nbdg, by Thermo Fisher Scientific (2 mentions)
Mitotracker green, by Thermo Fisher Scientific (2 mentions)
Omnicyt flow cytometer, by Cytognos (1 mentions)
2 deoxyglucose 2 nbdg, by Thermo Fisher Scientific (1 mentions)
4 protocols using intracellular glutathione detection assay kit
1
Hemolymph Cell Viability Assay
2
Metabolic Profiling of Activated NKT Cells
3
Metabolic Profiling of Activated NKT Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To measure metabolic parameters, activated NKT cells (1 × 105) were incubated with different reagents as indicated in the figure legends. To measure mitochondrial mass and potential, cells were incubated with 30 nM MitoTracker™ Green (Invitrogen) and 60 nM of tetramethylrhodamine methyl ester perchlorate (TMRM) (Invitrogen), respectively, for 30 min at 37°C in RPMI 1640 complete media. Mitochondrial ROS levels were measured by incubating cells in 2.5 μM MitoSOX (Invitrogen) for 30 min at 37°C in RPMI 1640 complete media. To measure total cellular ROS, activated NKT cells were incubated with 1 mM 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) (Invitrogen) in RPMI complete media for 30 min at 37°C. To measure glucose uptake, cells were incubated in 2-(N-(7-nitrobenz-2-oxa-1,3-diaxol-4-yl) amino)-2-deoxyglucose (2-NBDG) (Invitrogen) (20 μM) for 1 h at 37°C in glucose-free RPMI 1640 media containing 10% dialyzed FBS. To measure GSH, cells were stained using an intracellular glutathione detection assay kit (Abcam) for 20 min at 37°C in RPMI 1640 complete media. Cells were stained for surface antigens and acquired on a FACS Canto II (BD Biosciences).
4
Metabolic Profiling of Activated NKT Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To measure metabolic parameters, activated NKT cells (1 x 10 5 ) were incubated with different reagents as indicated in the fig. legends. To measure mitochondrial parameters, cells were incubated with 60 nM of the potentiometric dye tetramethylrhodamine methyl ester perchlorate (TMRM) (Invitrogen), 30 nM MitoTracker TM Green (Invitrogen), and 2.5 µM MitoSOX (Invitrogen) for 30 min at 37˚C in RPMI 1640 complete media. To measure total cellular ROS, 1 x 10 5 activated NKT cells were incubated with 1 mM 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) (Invitrogen) in RPMI complete media for 30 minutes at 37 C. To measure glucose uptake, cells were incubated in 2-(N-(7-nitrobenz-2-oxa-1,3-diaxol-4-yl) amino)-2deoxyglucose (2-NBDG) (Invitrogen) (20 µM) for 1 h or as indicated at 37°C in glucose-free RPMI 1640 media containing 10% dialyzed FBS. To measure GSH, cells were stained using an intracellular glutathione detection assay kit (Abcam) for 20 min at 37°C in RPMI 1640 complete media. Cells were stained for surface antigens and acquired on a FACS Canto II (BD Biosciences).
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!