InCellGen™ MitoTracker Green£¬IC-1559

Description

InCellGen™ MitoTracker Green is the same molecule to the MitoTracker Green FM (M7514, ThermoFisher). It is green-fluorescent mitochondrial stain. Unlike other MitoLite probes, InCellGen™ MitoTracker Green appears to localize to mitochondria, much less depending on mitochondrial membrane potential. The dye stains live cells, but it is not well-retained after aldehyde fixation.

Protocol

1.Prepare 1 mM InCellGen™ MitoTracker Green stock solution

2.Prepare 20-200 nM InCellGen™ MitoTracker Green staining solution

3.Remove the growth media from the cells

4.Add InCellGen™ MitoTracker Green staining solution to cells

5.Incubate at 37¡ãC for 30 minutes

6.Wash cells and replace with 1x Hanks and 20mM Hepes Buffer (HH buffer)

7.Observe cells using a fluorescence microscope with FITC filter set

Note: Bring the dye at room temperature before starting the experiment.

PREPARATION OF STOCK SOLUTION

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 ¡ãC after preparation. Avoid repeated freeze-thaw cycles.

InCellGen™ MitoTracker Green stock solution:

Dissolve one vial InCellGen™ MitoTracker Green (50 ug) in 74 uL high-quality, anhydrous dimethylsulfoxide (DMSO) to make 1 mM stock solution.

Note: Keep the stock solution frozen at ¡Ü¨C15¡ãC and protected from light.

PREPARATION OF WORKING SOLUTION

InCellGen™ MitoTracker Green staining solution:

Dilute 1 mM InCellGen™ MitoTracker Green stock solution to the final working concentration in HH buffer. The working concentration can be in the range of 20¨C200 nM.

SAMPLE EXPERIMENTAL PROTOCOL

Staining adherent cells:

1.Grow cells to reach the desired confluency.

2.Remove the growth media from the cells.

3.Add InCellGen™ MitoTracker Green staining solution to each well.

4.Incubate at 37¡ãC for 30 minutes.

5.Wash cells and replace with 1x Hanks and 20mM Hepes Buffer (HH buffer).

6.Observe cells using a fluorescence microscope with FITC filter set.

Note: The staining protocols is good for Hela cell line and it may need to be optimized with the particular cell types.

Staining suspension cells:

1.Centrifuge cells to a pellet and aspirate the supernatant.

2.Resuspend the cells gently in InCellGen™ MitoTracker Green staining solution.

3.Incubate at 37¡ãC for 30 minutes.

4.Centrifuge the cells, remove supernatant and resuspend cells in fresh HH buffer.

5.Cells may be analyzed by flow cytometry (530/30 nm filter- FITC channel) or fluorescence microscopy (FITC filter set).

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