Can Propidium Iodide Be Fixed?

What does propidium iodide do?

Propidium iodide is used as a DNA stain in flow cytometry to evaluate cell viability or DNA content in cell cycle analysis, or in microscopy to visualize the nucleus and other DNA-containing organelles..

How does propidium iodide staining work?

Propidium iodide (PI) is a membrane impermeant dye that is generally excluded from viable cells. It binds to double stranded DNA by intercalating between base pairs. PI is excited at 488 nm and, with a relatively large Stokes shift, emits at a maximum wavelength of 617 nm.

How long can I keep fixed cells?

You can store them there for several years if needed. It gives very nice IF staining. Lately, i used cell cultures fixed in acetone and stored for 12 months in the -80°C and the stainings were very pretty using golgi staining, ER staining etc.

How do you dissolve propidium iodide?

To make a stock solution from the solid form, dissolve PI (MW = 668.4) in deionized water (dH2O) at 1 mg/mL (1.5 mM) and store at 4°C, protected from light. When handled properly, solutions are stable for at least 6 months.

How long can you store fixed cells for flow cytometry?

3 daysOnce fixed, cells can be stored for a few days (try not to exceed 3 days). Most of surface antibodies would either not recognize or suboptimally recognize fixed epitopes, that’s why you want to work fresh. For cytoplasmic epitopes, the antibodies are developed based on the requirement to recognize fixed epitopes.

Is propidium iodide toxic?

CONSIDERED A HAZARDOUS SUBSTANCE ACCORDING TO OSHA 29 CFR 1910.1200. Harmful if swallowed. Possible risk of irreversible effects. Irritating to eyes, respiratory system and skin.

Does propidium iodide expire?

The expiration date is indicated on the vial label. Propidium iodide (PI) is a fluorescent dye that intercalates into double-stranded nucleic acid. It is excluded from viable cells, but can penetrate cell membranes of dead or dying cells. … The fluorescence emission maximum for DNA-bound PI is about 615–620 nm.

What color is propidium iodide?

redPropidium iodide (PI) is a popular red-fluorescent nuclear and chromosome counterstain. Since propidium iodide is not permeant to live cells, it is also commonly used to detect dead cells in a population. PI binds to DNA by intercalating between the bases with little or no sequence preference.

Where does propidium iodide stain localize in the cell?

PI can only cross compromised bacterial membranes and is therefore considered to be an indicator of membrane integrity. It stains DNA and RNA inside of dead cells or the ones with reversibly damaged membranes.

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.

Can you over fix cells?

Longer fixation times are sometimes necessary when dealing with tissues, but this is only so that the fixative can fully penetrate the tissue. Over-fixation can mask antibody epitopes, and reduce antibody accessibility. In addition, longer fixation with PFA usually increases tissue autofluorescence.

Can you sort fixed cells?

Preserving high quality RNA for post-cell-sort sequencing in fixed cells can be achieved using a zinc-buffer fixation protocol.