BCECF is the most widely used fluorescent pH sensor, its pKa (6.98) is near physiological pH and allows the detection of cytosolic pH change with high sensitivity. The excitation spectrum of the dye undergoes a slight shift during pH change, while the wavelength of the emission maximum remains unchanged. The pH is determined ratiometrically by the relative fluorescent intensities at 535 nm when the dye is excited at 439 nm and 505 nm respectively. At low pH, BCECF is weakly fluorescent, however, fluorescence increases with increasing pH.
Generic loading protocol for BCECF:
mammalian cells can be loaded by incubation with the membrane permeant
BCECF AM. Inside the cell, nonspecific esterases hydrolyze the nonfluorescent AM ester into
the fluorescent, pH-sensitive indicator. The low
leakage rate of the negatively charged BCECF and the small intracellular
volume results in the higher intracellular concentration compared to the extral incubation concentration. BCECF is typically less susceptible to intracellular compartmentalization than calcium indicators.
1. Prepare cells in suspension
2. Dilute a1 mM aliquot of AM ester stock solution 100-500 fold into a physiological saline buffer (HBSS for example). It is recommended to use the minimum concentration of AM ester necessary to obtain an adequate signal (0.1 μM may be sufficient). The loading medium needs to be free of amino acids or buffers containing amines, to ensure there is no interference with hydrolosis of AM esters.
3. Add one-one volumes of aqueous AM ester dispersion and cell suspension. Incubate for 15–60 minutes at 4°C to 37°C.
4. Wash the cells with fresh culture medium 1-3 times.
The general protocol has also been aplied to tisue samples such as rat arteires and salivary glands, rabbit kidney and gastric glands. Typical applications involves mounting the tissue sample in a perfusion chamber and adding 1-5μM of BCECF to the perfusate for between 10-70 minutes. An wash withunmodified perfusate is then performed.
Learn more about BCECF
A group from Merck's Department of Exploratory Sciences and Screening presented a poster evaluating Asante Natrium Green-2 in a 1536 well voltage -gated sodium channel Assay.
Evaluation of the Sodium Sensing Dye Asante Natrium Green 2 in a Voltage-gated Sodium Channel Assay in 1536-well Format
Gregory T. O'Donnell, Kelli Solly, Carissa Quinn, Brian Squadroni, Eric Johnson, Jeffrey Hermes, and Michael Finley.
Department of Exploratory Sciences and Screening Merck Research Laboratories 140-154 Wissahickon Ave, North Wales, PA 19454, USA
High-throughput screening (HTS) of voltage-gated sodium channels has required the use of indirect measurements of channel activity partly due to the lack of a robust sodium sensitive dye. One popular screening method utilizes a dye that responds to changes in the membrane potential that results from sodium channel opening. Another approach, atomic absorption spectroscopy, makes use of surrogate ion transport through the voltage-gated ion channel of interest, substituting lithium for sodium. While these assays have been shown to be amenable to high-throughput screening, the direct measurement of sodium flux in an HTS-friendly read-out would be beneficial in lead identification efforts. Herein we describe a 1536-well FLIPR screening assay for antagonists to a voltage-gated sodium channel expressed in human embryonic kidney cells (HEK293) using a sodium sensing dye, Asante Natrium Green 2 (ANG-2, Teflabs). Addition of 60 μM of the site 2 agonist veratridine induced a signal-to-background ratio (S/B) of 1.3-1.6 fold (control wells versus wells treated with an IC100 of tetracaine). The assay was benchmarked against three known voltage-gated sodium channel blockers, tetracaine, flecainide, and mexilitene and exhibited acceptable sensitivity with IC50s of 2.7, 15.3, and 29 uM, respectively. The assay was then used to screen a library of 27,978 small molecules to assess the performance under screening conditions and compared to results using the same compound set screened with a membrane potential dye (Blue component A, Molecular Devices). The sodium dye assay gave robust statistics with Z' values averaging 0.71 and S/B averaging 1.58-fold over a 48 plate screening run. While the sodium dye showed good internal consistency (R2 = 0.80 when plotting duplicate data for each compound against each other), the membrane potential and sodium dye assays showed a weaker correlation with an R2 = 0.46. Using a 40% activity cut-off, 2237 compounds showed overlapping activity in both assays. However, each assay identified a large number of unique hits with 886 sodium dye only hits and 1186 membrane potential dye only hits. Data on the same compounds obtained from the IonWorks Quattro (Molecular Devices) system revealed that the sodium dye identified 39.3 % of all the electrophysiology positives, while the membrane potential dye identified 39.4 %. However, the sodium dye detected 129 electrophysiology actives that the membrane potential dye missed, while the membrane potential dye identified 130 compounds missed in the sodium dye assay. While neither assay appears capable of identifying all the potential inhibitors that may be uncovered using an electrophysiology read-out, both dyes provided robust read-outs in 1536-well format and would allow for the screening of large compound libraries. Additionally, the use of both membrane potential and sodium dyes in an HTS strategy may prove beneficial as both identify unique hits.
A total of 27,978 compounds were screened in the three different assays. The Venn diagram shows the hit distribution and overlap of those hits. Although the overall hit rates at 40% inhibition or greater for the sodium and membrane potential dye assays were similar (13.5% and 14.6%, respectively), each assay identified large numbers of assay specific hits. Combining the sodium and membrane potential assay hits, only 47% of all the electrophysiology hits were identified in the FLIPR screens.