Flow cytometry

Flow cytometry is an already approved method for quantitative and functional analysis of cells addicted to cell size, granularity and fluorescence intensity. A wide range of applications can be achieved by using flow cytometry (e.g. cell cycle analysis, apoptosis, membrane potential, etc.). In the present study flow cytometry was used for the quantitative analysis of intracellular ROS accumulation as well as determination of mitochondrial mass. All measurements were performed using the BD FACSort (Becton Dickinson). Fluorescence was measured using the appropriate filters for the respective fluorochromes. Data were analysed by the CellQuest software (Becton Dickinson) and WinMDI software (version 2.9). Suitable solutions, like sheath fluid (BD FACSFlowTM), cleaning (BD FACSClean) and rinsing solution (BD FACSRinse), were purchased from Becton Dickinson.

2.2.2.1. Determination of cellular ROS DICHLOROFLUORESCIN DIACETATE

2´,7´-Dichlorofluorescin diacetate (DCFH-DA), a derivative of fluorescein, is a cell-permeant indicator for free intracellular reactive oxygen species (ROS). The reduced and acetylated form (DCFH-DA) is non-fluorescent and able to pass the cell membrane. Cleavage of the acetate group by intracellular esterase yields a non-fluorescent charged form that is much better retained in the cell (DCFH). This compound is trapped inside the cells and oxidised to the fluorescent compound DCF depending on the cellular oxidation status. 300,000 cells were plated on 6 cm culture plates the day before the experiment. Cells were incubated with 2 µM DCFH-DA in DMEM without FCS for 45 min at 37°C in the incubator protected from light. Cells were harvested, resolved in 300 µl PBS(-) and transferred to round bottom polypropylene tubes (Greiner Bio-One GmbH). Fluorescence intensity was measured at an excitation wavelength of 488 nm and emission wavelength of 530 nm (FL-1). A number of approximately 10,000 cells was analysed for each sample. Mean fluorescence intensity (MFI) was measured for at least three independent experiments.

DCFH-DA solution

2 mM DCFH-DA

in ethanol

MITOSOXTM RED SUPEROXIDE INDICATOR

To investigate the production of cellular ROS in the c-myc/Ha-ras-transformed fibroblasts the MitoSoxTM Red superoxide indicator was used. After transfection with the lentivirus for c-myc

and Ha-ras the transformed cells express VENUS. The strong fluorescence of VENUS

makes it impossible to use DCFH-DA for the detection of cellular ROS. The non-fluorescent MitoSoxTM Red is cell-permeant and rapidly targeted to the mitochondria. There it becomes oxidised by superoxide and exhibits a red fluorescence. 300,000 cells were plated on 6 cm culture plates the day before the experiment. Cells were protected from light and incubated with 5 µM MitoSoxTM Red in DMEM without FCS for 30 min at 37°C. Cells were harvested, resolved in 300 µl PBS(-) and transferred to round bottom polypropylene tubes (Greiner Bio- One GmbH). Fluorescence intensity was measured at an excitation wavelength of 488 nm and emission wavelength of 580 nm (FL-2). A number of approximately 10,000 cells was analysed for each sample. MFI was measured for at least three independent experiments.

MitoSoxTM Red solution 5 mM MitoSoxTM Red in DMSO

2.2.2.2. Quantification of mitochondria in cells MITOTRACKER® GREEN AND RED

The Mitotracker® Probes contain a mildly thiol-reactive chloromethyl moiety and selectively label mitochondria in living cells. It is predicted that Mitotracker® Green and Mitotracker® Red can be used to measure mitochondrial mass independent of mitochondrial membrane potential (MMP) 242. In the present study both agents were used to determine the mitochondrial mass in non-transformed and c-myc/Ha-ras-transformed fibroblasts. Since the transformed cells exert strong fluorescence due to VENUS expression, Mitotracker® Red was chosen. 300,000 cells were plated on 6 cm culture plates the day before the experiment. Cells were protected from light and incubated with 20 nM Mitotracker® Green or with 100 nM Mitotracker® Red in DMEM without FCS for 25 min at 37°C. Cells were harvested, resolved in 300 µl PBS(-) and transferred to round bottom polypropylene tubes (Greiner Bio-One GmbH). Mitotracker® Green fluorescence intensity was measured at an excitation wavelength of 448 nm and an emission wavelength of 516 nm (FL-1). Mitotracker® Red fluorescence intensity was measured at an excitation wavelength of 488 nm and an emission wavelength of 599 nm (FL-2). A number of approximately 10,000 cells was analysed for each sample. MFI was measured for at least three independent experiments.

Mitotracker® Green solution Mitotracker® Red solution

1 mM Mitotracker® Green 1 mM Mitotracker® Red

in DMSO in DMSO

NONYL ACRIDINE ORANGE

Alternatively, mitochondrial mass was measured using a second experimental setting by staining cells with nonyl acridine orange (NAO). NAO is well retained in the mitochondria and its uptake is reported to be independent from mitochondrial membrane potential (MMP) 287. 300,000 cells were plated on 6 cm culture plates the day before the experiment. Cells were protected from light and incubated with 10 nM NAO in PBS(+) without FCS for 20 min at 37°C. Cells were harvested, resolved in 300 µl PBS(+) and transferred to round bottom polypropylene tubes (Greiner Bio-One GmbH). Fluorescence intensity was measured at an excitation wavelength of 448 nm and an emission wavelength of 519 nm (FL-1).

Mitochondrial mass was normalised to the content of endoplasmic reticulum (ER). Therefore cells were stained with the ER-specific dye ER-TrackerTM Red. The stain consists of the green fluorescent BODIPY® TR dye and glibenclamide. Glibenclamide binds to the receptors of ATP-sensitive K+-channels which are prominent on the ER. Cells were protected from light and incubated with 1 µM ER-TrackerTM in PBS(+) with 5% FCS for 20 min at 37°C. Fluorescence intensity was measured at an excitation wavelength of 448 nm and an emission wavelength of 511 nm (FL-1). A number of approximately 10,000 cells was analysed for each sample. MFI for NAO and ER-TrackerTM was measured for at least three independent experiments. The estimated MFI for NAO was normalised to the MFI for ER- TrackerTM.

NAO-solution ER-TrackerTM

1 µM Acridine Orange 10-NONYL 1 mM ER-TrackerTM

in ethanol in DMSO