How do you make fluorescein diacetate solution?
FDA stock solution is prepared by dissolving 5 mg of fluorescein diacetate in 1 ml acetone, and sucrose may be added for live cell viability testing. FDA stain must be kept in the dark at 4°C or it will spoil.
How Fluorescein diacetate works?
Fluorescein diacetate (FDA) is a cell-permeant esterase substrate that can serve as a viability probe that measures both enzymatic activity, which is require to activate its fluorescence, and cell-membrane integrity, which is required for intracellular retention of their fluorescent product.
How does FDA staining work?
The principle of FDA staining is based on the ability of FDA to pass through the hydrophobic cell membrane of viable mycobacteria, followed by rapid hydrolysis of FDA by cell esterases [15], [16], which are assumed to be present only in live cells.
How does propidium iodide staining work?
Propidium iodide (or PI) is a fluorescent intercalating agent that can be used to stain cells and nucleic acids. PI binds to DNA by intercalating between the bases with little or no sequence preference. PI also binds to RNA, necessitating treatment with nucleases to distinguish between RNA and DNA staining.
How do you dilute fluorescein?
First, you may prepare a solution at 1mg/ml (2.6 mM) in molecular grade water, which you can then dilute to generate a 100 uM stock solution. Finally, we recommend preparing a working solution at 1 uM, which you can add directly to the PCR mix.
How does FDA prepare stock solutions?
Prepare a 5 mg/mL FDA stock solution in acetone. Dilute the stock solution ~1:100 in either H2O or PBS.
Is fluorescein cell permeable?
The fluorescent dye fluorescein is membrane-permeable due to derivatization to FDA and can enter cells without specific uptake mechanisms.
Can I use PE and PI together?
Propidium iodide (PI) is a membrane impermeant dye that is generally excluded from viable cells. Because of these spectral characteristics, PI can be used in combination with other fluorochromes excited at 488 nm such as fluorescein isothiocyanate (FITC) and phycoerythrin (PE).
How do you make propidium iodide solution?
PI Stock Solution (1mg/ml or 1.5 mM): To prepare, add 1 mg PI (Propidium Iodide) to 1 ml distilled water. Store stock solution at 4 ºC (or aliquot and store at -20 ºC), protecting from light. When handled properly, solutions are stable for many years.
Do you dilute fluorescein?
Do not mix or dilute with other solutions or drugs. Flush intravenous cannulas before and after drugs are injected to avoid physical incompatibility reactions.
What is fluorescein sodium?
Similar to ICG, FS is a synthetic organic fluorescent dye. The FDA has approved its use for diagnostic fluorescein angiography or angioscopy of the retina and iris vasculature. Its major excitation peak is between 465 and 490 nm and the fluorescence emission band is from 510 to 530 nm.