求使用MitoSOX检测线粒体ROS产生的protocal.

fansimiao@yahoo

准备使用mitosox检测凋亡过程中ROS的产生，求详细的protocol.

niwanmao

fansimiao@yahoo 发表于 2011-6-10 07:29

                               
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准备使用mitosox检测凋亡过程中ROS的产生，求详细的protocol.

这个不是吗？同样也适用于流式（红光）。http://probes.invitrogen.com/media/pis/mp36008.pdf

Reagent Preparation
Prepare 5 mM MitoSOX™ reagent stock solution.
Dissolve the contents (50 μg) of one vial of MitoSOX™ mitochondrial superoxide indicator (Component A) in 13 μL of dimethylsulfoxide (DMSO) to make a 5 mM MitoSOX™ reagent stock solution

Labeling Live Eukaryotic Cells
This protocol was developed using live bovine pulmonary epithelial (BPAE) cells, MRC5 human lung fibroblasts, and mouse 3T3 fibroblasts adhering to coverslips, but can be adapted for use with other cell types. Recommendations for experimental protocols should be used as a starting point, and optimal labeling conditions should be determined empirically.

1.1 Prepare 5 μM MitoSOX™ reagent working solution.
Dilute the 5 mM MitoSOX™ reagent stock solution (prepared above) in HBSS/Ca/Mg or suitable buffer to make a 5 μM
MitoSOX™ reagent working solution.
Note: The concentration of the MitoSOX™ reagent working solution should not exceed 5 μM. Concentrations exceeding 5 μM
can produce cytotoxic effects, including altered mitochondrial morphology and redistribution of fluorescence to nuclei and the
cytosol.
1.2 Load cells. Apply 1.0–2.0 mL of 5 μM MitoSOX™ reagent working solution (prepared in step 1.2) to cover cells adhering
to coverslip(s). Incubate cells for 10 minutes at 37˚C, protected from light.
1.3 Wash cells. Wash cells gently three times with warm buffer.
1.4 Prepare cells for viewing. Stain cells with counterstains as desired and mount in warm buffer for imaging。