Dyes for PFA 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.

EMA (Ethidium Monoazide):

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.

Preparation:

  • 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.

 

Spectral Properties:

  • 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 a 488 nm laser, (excitation max = 510 nm) with detection in the PECy5 or PE-Texas red channels (emission max = 600 nm).
  • Include an EMA compensation control when using in multicolour flow cytometry applications

 

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