Dear Thirumala!

Please You look at the following article:

Article 17β-estradiol modifies human spermatozoa mitochondrial funct...

Detection of mitochondrial superoxide anion

In order to estimate the amount of superoxide anion produced in spermatozoal mitochondria, we used MitoSOX Red fluorochrome (Molecular Probes). The analysis was performed according to the method described by Koppers et al. [27] with the use of confocal microscope and flow cytometer. MitoSOX Red stock solution (5 mM diluted in DMSO) was added to cell suspension (20 × 106 cells per mL) to give the final concentration of 2 μM. Cells were incubated for 15 min in darkness at 37 °C and afterwards washed twice with F-10 medium (5 min at 600 × g). Microscopic observation was made under LSM 510 confocal microscope (Carl Zeiss GmbH, Germany). MitoSOX Red fluorescence was measured using flow cytometer FACSCalibur (Becton–Dickinson, USA). The results were expressed as the percentage of MitoSOX positive cells and as mean fluorescence intensity of MitoSOX positive cells.

Sperm vitality and phosphatidylserine membrane translocation

To determine phosphatidylserine membrane translocation (PST) from the inner to the outer layer of the plasma membrane, the annexin-V labeled with fluorescein (AnV-FLUOS) (Roche Molecular Diagnostics, Darmstadt, Germany) was used. Simultaneously, to distinguish between viable and dead spermatozoa the propidium iodide (PI) staining was used, in the final concentration of 0.125 μg/L (Sigma-Aldrich, St. Louis, MO). Double staining was conducted according to manufacturer’s recommendations.

Flow cytometry

The fluorescence signals of labeled spermatozoa were analyzed by flow cytometer FACSCalibur. 10 000 cells were examined for each experiment. The fluorescence of An-V-FLUOS and PI was excitated by argon laser (488 nm) and emission of An-V-FLUOS was measured in the FL1 channel (515–545 nm), while the red fluorescence of PI was detected in the FL3 channel (650 nm). The fluorescence of MitoSOX Red was analyzed in the FL2 channel (561–603 nm). The emission of JC-1 monomers and aggregates was measured in the FL1 channel (515–545 nm) and FL2 channel (561–603 nm), respectively. All data was collected and analyzed using CellQuest Pro software (v.5.2.1) (Becton–Dickinson).

Thank you dear Alexandr Chernov for your answer. I am actually looking for the pictures of fluorescence microscope (FM). How the staining pattern is observed in FM. yes in FC we are getting the results.

Sir beacuse the reduce dye binds to the nucleic acod present in nucleus more the free radical more will be reduction of dye and subsequently more flourescent will be produced on binding to nucleic acid