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)
 Preparation:
  • 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.
 Procedure:
  • 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.
Spectral Properties:
  • 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).

Fluorescent emission profile of Propidium Iodide and spectral overlap with commonly used fluorophores when excited at 488nm.

7-AAD (7-Aminoactinomycin-D):
Preparation:
  • 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.
 Procedure:
  • 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.
 Spectral Properties:
  • 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.

Fluorescent emission profiles of 7-amino actinomycin D when and other commonly used fluorophores when excited at 488nm

 

DAPI (4′, 6-Diamidino-2-Phenylindole, Dihydrochloride):
Preparation:
  • 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.
Procedure: 
  • 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.
Spectral Properties:
  • 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.

Emission spectrum of DAPI compared to other commonly used fluorophores

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