Fixable Viability Dyes for Flow Cytometry

Figure 1. Principle of the LIVE/DEAD Fixable Viability Dead Cell Stains.The cell-impermeant, amine-reactive dye only binds to the surface of the live cell, resulting in very dim fluorescence. The dye can penetrate the cell membrane in dead cells and will bind to internal proteins, resulting in very bright fluorescence.

Figure 2. Retention of LIVE/DEAD Fixable Viability Stains after fixation. The LIVE/DEAD Fixable Red (615) Viability kit for 488 and 561 nm excitation was used to differentially stain a mixture of live (left peak) and heat-treated Jurkat cells (right peak). Cells in (A) were not fixed; cells in (B) were fixed in 3.7% formaldehyde following staining. Samples were analyzed by flow cytometry using 488 nm excitation and ~615 nm emission.

Figure 3. Cells stained with PI do not retain staining pattern after fixation. Propidium iodide (PI) was used to differentially stain a mixture of live (left peak) and heat-treated Jurkat cells (right peak). Cells in (A) were not fixed; cells in (B) were fixed in 3.7% formaldehyde following staining. Samples were analyzed by flow cytometry using 488 nm excitation and ~617 nm emission.

Step 1: Stain cells with LIVE/DEAD reagent
Protocol for LIVE/DEAD cell staining.
Minimize FBS or BSA or protein buffer to <1%

Step 2: Use a fixation and/or permeabilization buffer kit if staining with intracellular markers or to persevere stained cells. Do not use a fixation agent before the LIVE/DEAD reagent.

Step 3: Stain cells with flow cytometry antibodies.
Combinations of dyes and clones, and reagents can be created on the Invitrogen Flow Cytometry Panel Builder.
Protocol for intracellular staining for flow cytometry.

Figure 5. Live cells will have less fluorescence since LIVE/DEAD dye will bind to cell surface proteins. Dead cells will be the far-right population and exhibit bright signal as LIVE/DEAD dye goes through damaged cell membranes and binds to internal proteins. Middle population is structural degradation of cells and will incorporate less dye than dead cells. Jurkat cells were incubated with LIVE/DEAD Fixable Lime (506) Viability kit for 405 nm excitation for 20 mins and then fixed. Dead cells were induced by heat treatment.