Protocols

T. J. Fetterhoff, S.P. Holland, and K. J. Wile, Boehringer Mannheim Corp., Research & Development, Indianapolis, IN, USA. Cytometry Supplement 6: 27 (1993).

Cell lysis and or fixation have become common practice in flow cytometric sample preparation. A procedure has been developed that allows detection of cells that were non-viable prior to cell fixation. This technique overcomes the problems of artifacts caused from non-specific uptake of antibodies by non-viable cells at the time of staining. 7-Amino-Actinomycin D (7-AAD) is used to identify non- viable cells during immunostaining. Following several washes, cells are lysed or fixed in solutions that contain molar excess of the non-fluorescent Actinomycin D (AD). AD competes with 7-AAD for binding sites to dsDNA and inhibits further uptake by 7-AAD. Furthermore, 7AAD exhibits spectral characteristics enabling fluorescence separation from FITC as well as PE. This procedure allows cell fixation to inactivate infectious agents and eliminates artefactual staining by non-viable cells. © 1993 Wiley-Liss, Inc.

Materials

Protocol

  1. Resuspend labeled cells in 100 m l PBS that contains 20 mg 7-AAD; incubate for 30 minutes at 4°C.
  2. Centrifuge and resuspend pellet in 100 ml of PBS that contains 0.5% para-formaldehyde and 50 mg actinomycin-D; incubate 30 minutes at 4°C
  3. Centrifuge and resuspend pellet in PBS; hold at 4°C in the dark.

References

  1. Schmid I, Uittenbogaart CH, Krall WJ, Braun J and Giorgi JV. Dead cell discrimination with 7-amino-actinomycin D in combination with dual color immunofluorescence in single laser flow cytometry. Cytometry 13:204-208, 1992.
  2. Fetterhoff TJ, Holland SP, and Wile, KJ. Fluorescent detection of non-viable cells in fixed cell preparations. Cytometry 14 (Suppl. 6):27, 1993.

ADHERENT CELL DNA STAINING

Only use if the other protocols fail. The samples are stored before staining, but it's more complicated.