How do you use 7-AAD in flow cytometry?
To adjust flow cytometer settings for 7-AAD, add 5 – 10 μL of 7-AAD staining solution to a control tube of unstained cells. Mix gently and incubate for 30 minutes at 4 °C in the dark. Determine 7-AAD fluorescence (using the FL-2 or FL-3 channel) with a FACScan™ instrument.
Can you fix 7-AAD?
7-AAD appears to be generally excluded from live cells, but can be used with cells that have been fixed and permeabilized. 7-AAD has been used for cell cycle analysis by flow cytometry.
How does propidium iodide staining work?
Propidium iodide (PI) is a cell-impermeant DNA binding dye that can be used to stain cells and nucleic acids. PI binds to DNA by intercalating between the bases with a stoichiometry of one dye per 4-5 base pairs of DNA. As a membrane impermeant dye, PI is generally excluded from viable cells.
What channel is 7-AAD?
When excited at 488 nm, 7-AAD is detected in the red fluorescence channel commonly used for R-phycoerythrin (PE)-Cy®5 tandem dye detection, with minimal spectral overlap into the yellow fluorescence channel commonly used for PE detection.
How do you use 7 AAD stain?
Dissolve 1 mg of 7- AAD powder by adding 50 microliters of absolute methanol directly to the vial. Mix well and add 950 microliters of 1 X PBS with Ca2+ and Mg2+ to achieve a concentration of 1 mg/ml. Store solution tightly closed and protected from light at 4°C.
What is 7-AAD stain?
7-AAD (7-amino-actinomycin D) is a ready-to-use solution for the exclusion of nonviable cells in flow cytometric analysis. 7-AAD can be used in place of PI (propidium iodide).
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.
What is FACS analysis?
Fluorescence activated cell sorting (FACS) analysis is a derivative of flow cytometry that proceeds in a slightly different direction. The primary objective of FACS is to physically sort a heterogeneous cell sample into separate populations. Isolated cells can then be used for further research.
What is the difference between FACS and flow cytometry?
Flow cytometry measures the properties of cells such as the number, size, and nucleic acid content of cells, while FACS separates cells into subpopulations from a heterogeneous mixture.