Hoechst 33342 / MTG / CMX Rosamine Protocol for Apoptosis

Poot, M. and Pierce, R.H. (1999) Detection of changes in mitochondrial function during apoptosis by simultaneous staining with multiple fluorescent dyes and correlated multiparameter flow cytometry. Cytometry 35; 311-317. Poot M and Pierce RH. Analysis of mitochondria by flow cytometry. Methods Cell Biol 2001;64:117-28. Poot M. Mulitparameter Analysis of Physiological Changes in Apoptosis. Current Protocols in Cytometry (2000) 9.15.1-9-9.15.7.

This protocol determines the number of live (Hoechst 33342 positive and MTG/CMXRos high) vs. early apoptotic (Hoechst 33342 positive and MTG/CMXRos low) cells and debris signals. In this protocol, three dyes are used. Hoechst stains all cells that contain DNA and resolves cells according to cell cycle stage; CMXRos is sensitive to changes in mitochondrial membrane potential; MTG reports the level of mitochondrial protein in cells. The reason for using MTG and CMXRos simultaneously is that this combination gives a better resolution between apoptotic (compromised mitochondria) and normal cells.

  1. Make the following stock solutions:
    • 1 mM Hoechst 33342 [cat # H-1399 Molecular Probes] in distilled water (do NOT use PBS, since phosphates will precipitate the dye)
    • 20 µM MitoTracker Green (MTG) [cat # M7514 from Molecular Probes, Inc.] in DMSO.
    • 20 µM MitoTracker red CMXRos [cat.# M-7512 from Molecular Probes, Inc.] in DMSO.
    Hoechst 33342 solution keeps in the refrigerator in the dark for weeks, MTG solution keeps in the freezer in the dark for weeks, CMXRosamine solution keeps in the freezer in the dark for weeks.
  2. Bring cells into suspension; preferably at a density around 2 million per mL.
  3. Add sequentially per mL cell suspension:
    • 10 µL Hoechst 33342
    • 1 µL MTG
    • 1 µL CMXRosamine
  4. Incubate at 37°C for 30 minutes.
  5. Cells should be analyzed on the flow cytometer as soon after staining as possible.