Mitotracker green

A.D. Presley, K.M. Fuller and E. A. Arriaga, MitoTracker Green labeling of mitochondrial proteins and their subsequent analysis by capillary electrophoresis with laser-induced fluorescence detection. J. Chromatogr.B, 793 (2003) 141-150. 

Fig-3 Fluorescence microscopy of Blastocystis revealing MitoTracker Green uptake of MLOs with DAPI colocalization. The MLOs are situated very close to the nucleus (large DAPI-stained structures) and are clustered at opposite poles of the cell... 

Pendergrass W, WolfN, Poot M (2004) Efficacy of MitoTracker Green and CMXrosamine to measure changes in mitochondrial membrane... 

Fig. 1. Cytogram of MitoTracker Green FM and CMXRosamine fluorescence (A) and cell-cycle histograms of normal (B) and compromised (C) cells from a culture of human lymphoblastoid cells that were treated with 1 mg/mL mitomycin C for 8 h at 37°C, harvested and stained according to Protocol 1. Apoptotic cells show a lower CMXRosamine and a higher MitoTracker Green FM fluorescence (A). The two right hand panels show the cell-cycle distributions, based on Hoechst 33342 fluorescence, of the normal and compromised cells.

Fig. 2. Cytogram of a combined assay for NAD(P)H levels (UV-excited blue autofluorescence) and mitochondrial membrane potential (CMXRosamine/ MitoTracker Green FM fluorescence). Signals representing normal and compromomised cells are labeled.

As with flow cytometry, multiparameter apoptosis assays may also be performed by confocal laser scanning microscopy (CLSM). Using the approach similar to that detailed above for flow cytometry, we have examined NADPH content, mitochondrial membrane potential (CMX Rosamine fluorescence), and mitochondrial mass (Mitotracker Green), by CLSM. Figure 3 shows an example of a typical multiparameter assay performed by confocal microscopy. 

In this protocol the UV excited blue fluorescence of Hoechst 33342 dye, which resolves cells according to the Gl, S, and G2 stage of the cell cycle, is combined with the green and red fluorescence of MitoTracker Green FM and CMXRosamine. 

Aliquot cell suspensions into 12 x 75 mm polypropylene tubes. Add 20 pL of 1 pM Hoechst 33342 and 1 pL each of 200 pM MitoTracker Green FM and CMXRosamine dye stock solutions into 1 mL of prewarmed cell suspension. Mix immediately by briefly vortexing at maximal speed. Incubate for 30 min. at 37°C in the dark or at subdued light. After staining, place tubes with cell suspensions in a melting ice bath (see Note 4). 

Set up and optimize the flow cytometer as in Basic Protocol 1. UV-excited (360 nm) blue fluorescence (collected with a 450 nm centered bandpass filter) from the Hoechst 33342 dye is proportional with cellular DNA content. To excite MitoTracker Green FM and CMXRosamine stained samples, the argon-ion laser should be tuned to the 488 nm line. To collect fluorescence from MitoTracker Green FM, use a bandpass filter centered around 530 nm for CMXRos use a longpass filter above 630 nm (see Notes 5-8). 

Keij, J. F., Bell-Prince, C., and Steinkamp, J. A. (2000) Staining of mitochondrial membranes with 10-nonyl acridine orange, MitoFluor Green, and MitoTracker Green is affected by mitochondrial membrane potential altering drugs. Cytometry 39(3), 203-210. 

Mitotracker Green FM Rhodamine 123 Fluorescein dextran conjugate LysoTracker Green Fluorescein Phalloidin Oregon Green 488 paclitaxel... 

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