CC2-DMPE and DiBAC4(3) ratiometric voltage reporter dyes were obtained from Invitrogen and used as per the standard protocol (Adams and Levin, 2013 (link)). Briefly, CC2-DMPE stock (5 mM) was dissolved 1:1,000 in 0.1× MMR and the embryos were incubated in the dark in this solution for at least 1 h followed by five washes with 0.1× MMR. DiBAC4(3) stock (1.9 mM) was dissolved 1:1,000 in 0.1× MMR and the CC2-DMPE-stained embryos were then incubated in the dark in this solution for at least 30 min washed thoroughly in 0.1× MMR followed by visualization under the microscope. An Olympus BX-61 microscope equipped with a Hamamatsu ORCA AG CCD camera and controlled by MetaMorph software (Molecular Devices), was used to collect images. ImageJ was used to quantify the fluorescence intensities of the CC2-DMPE: DiBAC signal along the red line across the image as indicated in the illustrations in Figures 4G , 9E ). Fluorescence values at each point along this line were used to plot graphs.
Cc2 dmpe
Manufactured by Thermo Fisher Scientific
Sourced in United States
CC2-DMPE is a chemical compound used in the preparation of liposomes and other lipid-based formulations. It functions as a structural lipid component that contributes to the formation and stabilization of these lipid-based systems.
Automatically generated - may contain errors
Lab products found in correlation
4 protocols using cc2 dmpe
1
Ratiometric Voltage Reporting in Xenopus
2
Ratiometric Analysis of Voltage Dynamics
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
CC2-DMPE and DiBAC4(3) voltage reporter dyes were obtained from Invitrogen and used as per the standard protocol, including dark-field and flat-field correction (Adams and Levin, 2012 (link)). Briefly, the use of two dyes with opposite emission profiles simultaneously provides an internal control and allows ratiometric normalization. CC2-DMPE stock (5 mM) was dissolved 1:1000 in 0.1× MMR and the embryos were incubated in dark in this solution for at least 1 h followed by washes with 0.1× MMR. DiBAC4(3) stock (1.9 mM) was dissolved 1:4000 in 0.1× MMR and the CC2-DMPE-stained embryos were then incubated in dark in this solution for at least 30 min followed by visualization under the microscope. An Olympus BX-61 microscope equipped with a Hamamatsu ORCA AG CCD camera, and controlled by Metamorph software (Molecular Devices), was used to collect signal. NIH Image J software was used to quantify the fluorescence intensities of the CC2-DMPE:DiBAC signal.
3
Voltage Sensor Probe Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A voltage sensor probe (VSP) set (CC2-DMPE and DisBAC2(3)), VABSC-1 (Voltage Assay Background Suppression Compound), and Pluronic®-F127 were purchased from Invitrogen (Carlsbad, CA, USA). BTX and TTX were purchased from Latoxan (Valence, France). Asante NaTRIUM Green-2 (ANG-2) was purchased from Interchim (Montluçon, France). All other reagents (Ponceau 4R) and solvents were obtained from Sigma-Aldrich (Saint-Louis, MO, USA) or Fisher Scientific (Waltham, MA, USA).
4
FRET-Based Membrane Potential Sensing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Voltage sensor probes, coumarin-labeled phospholipid CC2-DMPE (FRET donor) and oxonol dye DiSBAC2(3) (acceptor) were from Invitrogen. Cells (3×104 cells/well) in optical 96-well plates were loaded with 10 µM CC2-DMPE in FRET buffer (10 mM Hepes-NaOH, pH 7.4, 160 mM NaCl, 0.9 mM CaCl2, 1 mM MgCl2, and 10 mM glucose) containing 200 µg/ml Pluronic F-127 for 30 min, washed and incubated in 100 µl 10 µM DiSBAC2(3) in FRET buffer for 20 min. Tartrazine (1.2 mM final concentration) was added, and after 10 min, fluorescence measurements (λex of 420 nm; λem of 460 nm and 550 nm; 10-nm bandpass filters) were conducted in well mode at 37°C and 5% CO2. Gain was adjusted to yield similar baseline readings for each fluorophor at resting potential. Following baseline acquisition, 10 µl 0.82 M KCl, 1 mM BzATP or buffer control were injected while monitoring fluorescence. Following subtraction of signal without cells, the signal ratio (SR) before and at equilibrium after depolarization was calculated, and the response ratio (RR) derived as RR=SRdepol/SRpol.
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!