Dyes for Unfixed Samples
When live cell analysis is required, the following stains may be used to separate dead/dying cells from healthy cells. Dyes like PI/7-AAD and DAPI are not able to transit across intact cell membranes and are not fluorescent or have only weak fluorescence until intercalated between the DNA strands. This makes them excellent dead cell probes as they yield fluorescence once inside the cell.
For most flow cytometry experiments with these dyes, cells are stained with other fluorophores, using standard staining methods required for the primary assay, followed by the addition of the dead cell markers without a final wash step.
Note: All reagents below are available in working concentrations from various suppliers, but if you wish, they can be made from dry reagents at much less cost.
PI (Propidium Iodide):
- Reagents: PI-Sigma-Aldrich P4170), PBS, Sodium Azide (Sigma-Aldrich S8032)
- Prepare a stock solution of PI at 1 mg/mL in PBS containing 0.01% Sodium Azide (Undiluted stock is stable for up to 6 months at 4°C).
- Aliquot into 2-4 mL portions and store at 4 degrees wrapped in foil.
- Dilute an aliquot of the PI stock solution to 100 µg/mL.
- Add PI washed and stained cells ready for flow cytometric analysis.
- Add 1uL of PI stock solution per each 100 uL of staining buffer in your samples.
- Incubate cells in the dark for 5-15 mins – Do not wash.
- Acquire data on a flow Cytometer.
- Strong excitation at 488 nm (Blue laser), stronger excitation at 561 nm (yellow-green).
- Detected anywhere from 550 – 700 nm.
- Significant overlap between PE and PE-tandems to 700 nm.
- Minimal compensation requirements required between FITC/GFP and PI if data acquired at longer wavelengths (~650-700 nm).
Figure 1: Fluorescent emission profile of Propidium Iodide and spectral overlap with commonly used fluorophores when excited at 488nm.
- Reagents: 7-AAD (Sigma-Aldrich A9400), PBS, Sodium Azide (Sigma-Aldrich S8032), DMSO (D5897).
- Prepare a stock solution of 7-AAD at 1 mg/mL by dissolving 1.0 mg 7-AAD powder into 50 uL of DMSO.
- Add 950 uL of PBS containing 0.01% Sodium Azide
- Store in the fridge for 1 month in the dark.
- Add 1uL of 7-AAD stock solution to approximately 1 06 cells ready for analysis.
- Incubate cells in the dark for 15 mins at room temperature – do not wash.
- Acquire data on a flow Cytometer.
- 7-AAD is excited at 488 nm and emits at ~670 nm flow cytometer or can be detected between 600 and 750 nm.
- 7-AAD is less bright than PI, but gives good resolution between live and dead cells.
- There is also less spillover with PE conjugates making it easier to compensate from these detectors.
Figure 2: Fluorescent emission profiles of 7-amino actinomycin D and other commonly used fluorophores when excited at 488nm
DAPI (4′, 6-Diamidino-2-Phenylindole, Dihydrochloride):
- Reagents: DAPI (Sigma – 10236276001)
- Preparation of stock solution: Dissolve in de-ionized water to a final concentration of 1 to 5 mg/ml.
- Note:Do not use any buffers.
- Preparation of working solution: Dilute the stock solution with methanol to a final concentration of 1 μg/ml. The working solution is stable at 2 to 8 °C for about 6 months.
- Storage conditions:
- Stock solution (1 to 5 mg/ml) at -15 to -25 °C for 12 months.
- Working solution (1μg/ml) at 2 to 8 °C for about 6 months.
- Storage conditions:
- Prepare samples for flow cytometry.
- After the final wash step resuspend the cells in PBS with 1- 2% FBS and sodium azide containing 0.05-0.2 μ g/mL DAPI.
- The optimal concentration of DAPI for viability analysis may vary by cell type. Please titrate the reagent for your cell type to ensure good resolution without oversaturation.
- Do not add azide to the buffer if cells are being prepared for sorting.
- Incubate 5 minutes at room temperature. Do not wash.
- Acquire flow cytometry data.
- DAPI can be excited at 355, 375 or 405 nm with emissionmax at 460 nm (450/50 bandpass filter).
- DAPI cannot be used with fluorophores like Brilliant Violet 421, eFluor450, VioBlue Dye, V450 or Alexa Fluor® 405.
- Use of the violet laser for DAPI excitation leaves commonly used detectors for the blue and red lasers free with little expectations for compensation between colours (depending on fluors chosen.
Figure 3: Emission spectrum of DAPI compared to other commonly used fluorophores
Dyes for Fixed Samples
There are many more dead-cell discrimination dyes available with varied excitations and emissions for use in immunophenotyping and other assays. These include the amine reactive dye sets (made by all major suppliers) that can be used in either live or fixed cell assays.
Amine Reactive Dyes:
Amine reactive dyes are another class of molecules that selectively label dead versus live cells. Like classical DNA binding dyes, they are similarly reliant on cell permeability to enter the cell. Unlike DNA stains, these dyes irreversibly react with free amine groups of proteins both on the surface of or inside cells. The availability of the free amine groups on the cell’s interior versus exterior is much greater and therefore dead cells stain much more brightly in comparison to healthy cells. These dyes can also withstand harsh cross-linking reactions, making them very useful to measure viability at the time of fixation.
Figure 4: Amine reactive dyes bind only to the surface of live cells (left) and in lesser amounts when compared to cells that have lost membrane integrity (right). The porous membrane allows greater dye access to the free amines group of intracellular proteins which yields a brighter fluorescent signal when viewed on a flow cytometer.
These dyes have become more popular since first coming onto the market approximately 10 years ago and are available in an array of colours allowing for easy incorporation into any staining panel.
EMA (Ethidium Monoazide) Bromide:
Ethidium monoazide bromide is a fluorescent nucleic acid stain that covalently binds DNA after photolysis when exposed to UV light. Because this probe is relatively impermeant to live cells, it selectively labels DNA in dead vs. live cells making it useful in complex staining protocols involving fixation and further staining. This method is less widely used simply due to attrition and the popularity of the amine reactive dyes, but remains a cheap alternative for dead cell staining offering an additional choice for emissions not available with the amine reactive dyes i.e. PE-Texas red wavelengths.
- Reagents: EMA powder Molecular Probes: E1374 or Sigma-Aldrich: E2028
- Prepare EMA stock solution to 5mg/mL in DMSO and store as single use aliquots in a desiccator at -20 °C under foil or in light resistant tubes.
- Depending on the optimal working concentration for your current lot, add enough EMA (dilute accordingly) to achieve a final concentration of 0.5 – 5 μg/mL
- Titration will be necessary – Too much EMA can lead to extremely high background fluorescence leading to an underestimation of live cells….
- If using EMA at 0.5 μg/mL, add 10 μL of stock solution to 990 μl of FACS staining buffer.
- Add 1 μL of working solution/ 100 μL of final staining volume/ sample (stain cells in ~ 100-200 μL)
- Incubate cells from 10-15 minutes under a bright fluorescent lamp at distance of approximately 18 cm.
- Wash cells 2x with PBS
- Stain with extracellular flours and then fix as desired.
- EMA is very light sensitive to care must be taken to minimize exposure to light when making the stock solution, storage and up until ready to add to samples.
- EMA can be excited with 488 nm or 561 nm laser light (excitation max = 510 nm) with detection in the PECy5 (670nm) or PE-Texas (610) red channels (emission max = 600 nm).
- Include an EMA compensation control when using in multicolour flow cytometry applications
- EMA emits across a wide range of wavelengths (see spectrum below) and can potentially have large spillover values with fluors like PE-Cy5
Figure 5: EMA excitation at 488nm with an emission bandpass filter typical for PE-Texas Red centred at 610nm.
BD Biosciences: BD Horizon Fixable Viability (FVS) Reagents
Thermo Fisher: Fixable Viability Dyes for Flow Cytometry
Biolegend: All New Zombie Dyes (Blog Post)
ThermoFisher: Ethidium Monoazide Bromide
Perfetto S.P, et al. (2006) Amine reactive dyes: An effective tool to discriminate live and dead cells in polychromatic flow cytometery. Journal of Immunological Methods, 313, Issue 1-2, 30, pp 199-208. https://doi.org/10.1016/j.jim.2006.04.007
Kuonen, F., et al. (2010), Fc block treatment, dead cells exclusion, and cell aggregates discrimination concur to prevent phenotypical artifacts in the analysis of subpopulations of tumor-infiltrating CD11b+ myelomonocytic cells. Cytometry, 77A: 1082–1090. doi:10.1002/cyto.a.20969
Schmid I et. al, (2001), Simulatenous flow cytometric measurement of viability and lymphocyte subset proliferation, Journal of Immunological Methods, 247, Issue 1-2, pp. 175-186. https://doi.org/10.1016/S0022-1759(00)00323-9