7-AMINO-ACTINOMYCIN D STAINING OF DEAD CELLS FOR FLOW CYTOMETRY

7-Amino-actinomycin D (7-AAD) intercalates into double-stranded nucleic acids. It is excluded by viable cells but can penetrate cell membranes of dying or dead cells.

I. MATERIALS:

A.  7-Amino-actinomycin D (e.g., Cat #129935, Calbiochem, San Diego, CA)

B.  1 X PBS with Ca2+ and Mg2+

C.  Buffer: PBS (Ca2+ and Mg2+ free)
+2% newborn calf serum (or 0.2% BSA)
+0.1% sodium azide

7-AAD stock buffer:

For long-term storage, store unopened vials of 7-AAD in the freezer. Dissolve 1 mg of 7-AAD powder by adding 50 microliters of absolute methanol directly to the vial. Mix well and add 950 microliters of 1 X PBS with Ca2+ and Mg2+ to achieve a concentration of 1 mg/ml. Store solution tightly closed and protected from light at 4C. We have kept this solution for several months and have not observed loss in staining activity.

II. METHOD:

Stain your cells as outlined in the protocol for single color or dual-color staining with FITC and/or PE-labeled monoclonal antibodies.

After the last washing step resuspend your cells as usual in 1 ml of buffer for analysis. If you want to assess viability of your samples add 1-2 microliters of the 7-AAD stock solution to each tube and mix well. Keep the samples in this solution at 4C protected from light for approximately 20 minutes or until analysis on the flow cytometer.

NOTE: This method can now be used in combination with formaldehyde fixation of samples. Samples are first stained with 7-AAD, then fixed in 1% formaldehyde that contains 2-5 microliters/ml of actinomycin D (ref. Fetterhoff et al.); see attached protocol. 7-AAD can be used for dead cell exclusion on samples that are stained with PE (phycoerythrin)-conjugated antibodies, because the emission spectra of 7-AAD and PE can be easily separated on the flow cytometer.

Ref.: 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.

Fetterhoff TJ, Holland SP, and Wile, KJ. Fluorescent detection of non-viable cells in fixed cell preparations. Cytometry 14 (Suppl. 6):27, 1993.

Protocol for the use of actinomycin D (AD) on 7-AAD stained, formaldehyde-fixed samples

I. Materials:

A.  Actinomycin D ( C1) (AD, e.g., Cat# 102008, Boehringer Mannheim, IN)

B.  1 X PBS

C.  Sonicator

D.  Formaldehyde solution (see protocol for preparation of 2% stock solution)

 

II. Preparation of AD stock solution (1mg/ml):

To 1mg of AD powder add:

50 microliters of ice-cold absolute ETOH, vortex

950 microliters of 1 X PBS

Sonicate the resulting solution for 10 min at 4C; keep the solution overnight in the refrigerator at 4C, protected from light before using it.

Store solution at 4C protected from light.

Working dilution is 2-5 micrograms/ml.

 

III. Method:

Cells are first incubated with 7-AAD for approximately 20 min, spun down and washed once with 1 X PBS. Then, a 1% formaldehyde solution containing 2-5 microliters/ml of AD (F/AD) is quickly added to the cell pellet. Cells have to be stored in the cold protected from light and can be analyzed approximately 30 min after the addition of the F/AD solution. Cells are run on the flow cytometer in the F/AD solution. We have stored samples up to 3 days without any loss in the ability to discriminate dead from live cells.