RatioWorks™ BCFL, AM *Superior replacement for BCECF*
|Shipping||Standard overnight for United States, inquire for international|
|H-phrase||H303, H313, H333|
|Intended use||Research Use Only (RUO)|
|R-phrase||R20, R21, R22|
|Storage||Freeze (< -15 °C); Minimize light exposure|
|Excitation||Single: FITC, Ratio: AmCyan/FITC|
|Emission||Single: FITC, Ratio: AmCyan/FITC|
|Recommended plate||Black wall/clear bottom|
Fluorescence microplate reader
|Recommended plate||Black wall/clear bottom|
|Instrument specification(s)||Bottom read mode|
PREPARATION OF STOCK SOLUTIONS
Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles
Prepare a 10 to 20 mM stock solution of RatioWorks™ BCFL AM in high-quality, anhydrous DMSO.
Note: When reconstituted in DMSO, RatioWorks™ BCFL AM is a clear, colorless solution.
PREPARATION OF WORKING SOLUTION
On the day of the experiment, either dissolve RatioWorks™ BCFL AM in DMSO or thaw an aliquot of the indicator stock solution to room temperature. Prepare a RatioWorks™ BCFL AM working solution of 5 to 50 µM in a buffer of your choice (e.g., Hanks and Hepes buffer).
Note: The nonionic detergent Pluronic® F-127 can be used to increase the aqueous solubility of AM esters. In the staining buffer, the final Pluronic® F-127 concentration should be approximately 0.02%. A variety of Pluronic® F-127 products can be purchased from AAT Bioquest. Avoid long-term storage of AM esters in the presence of Pluronic® F-127.
Note: If your cells contain organic anion-transporters, probenecid (1-2 mM) may be added to the dye working solution (final in well concentration will be 0.5-1 mM) to reduce leakage of the de-esterified indicators. A variety of ReadiUse™ Probenecid products, including water-soluble, sodium salt, and stabilized solutions, can be purchased from AAT Bioquest.
SAMPLE EXPERIMENTAL PROTOCOL
The following is a recommended protocol for loading RatioWorks™ BCFL AM into live mammalian cells. This protocol only provides a guideline and should be modified according to your specific requirements.
- Prepare viable cells as desired.
On the next day, add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of the RatioWorks™ BCFL AM working solution into the cell plate.
Note: If your compound(s) interfere with the serum, replace the growth medium with fresh HHBS buffer (100 μL/well for 96-well plate or 25 μL/well for 384-well plate) before dye-loading.
- Incubate the dye-loaded plate in a cell incubator at 37 °C for 30 to 60 minutes.
- Replace the dye working solution with HHBS or buffer of your choice to remove any excess probes.
- Prepare the compound plates using HHBS or a buffer of your choice.
Run the pH assay as desired and simultaneously measure fluorescence using either a fluorescence microscope equipped with a FITC filter set or a fluorescence plate reader at Ex/Em = 490/535 nm cutoff 515 nm. For ratio measurements, monitor fluorescence at Ex/Em1 = 430/535 nm cutoff 515 nm and Ex/Em2 = 505/535 nm cutoff 515 nm.
Note: The compound addition is 50 μL/well (96-well plate) or 25 μL/well (384-well plate).
Note: Assays should be completed within 3 to 5 minutes after compound addition. However, a minimum of 8 minutes is recommended for data collection.
|Name||Excitation (nm)||Emission (nm)|
|RatioWorks™ BCFL Acid *Superior replacement for BCECF*||504||527|
|RatioWorks™ BCFL, SE||504||527|
|RatioWorks™ PH165, AM||463||641|
Authors: Lombardo, Tommaso
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