MitoTracker Red CMXRos, 50 μL

Product Information

Product Description/Introduction

MitoTracker Red CMXRos (Mitochondrial Red Fluorescent Probe) is an X-rosamine derivative with a molecular weight of 531.52, an excitation wavelength of 578 ± 3 nm and an emission wavelength of 599 ± 4 nm. The dye can directly enter living cells and locate on mitochondria and is able to covalently bind to mitochondria. The principle of specific labeling of mitochondria is that the probe skeleton with positive charge can be located on the negatively charged mitochondria, and the chloromethyl group on the probe can covalently bind to the free sulfhydryl group in the mitochondria , which plays the role of fluorescent labeling; the cell mitochondria can be labeled simply by incubating them with living cells for a certain period of time, and after labeling of the mitochondria, the mitochondria can be further processed through the fixation and perforation, which can be used for the subsequent multi-labeling experiments.

Storage and Shipping Conditions

Ship with wet ice; Store at -20 away from light, valid for 12 months.

Product Content

Assay Protocol / Procedures

1. Assay working solution preparation:

1.1. This product is in the form of a dry powder, which should be centrifuged briefly at high speed before use to ensure that it settles to the bottom of the tube. Provide your own cell grade DMSO.

1.2. Preparation of storage solution: Add 94 μL DMSO (cell level) into the tube, blow and mix well to fully dissolve the dye, that is to say, the mitochondrial fluorescent dye storage solution with a concentration of 1 mM (molecular weight information on the product label) is obtained, and then centrifuge briefly at a high speed to avoid the loss of reagents hanging on the wall. Store the storage solution at -20°C. It is recommended to store it in smaller sizes and avoid repeated freezing and thawing.

1.3. Preparation of assay working solution: If the assay is performed directly after staining, dilute the storage solution into mitochondrial assay working solution at a final concentration of 0.1-0.25 μM with biological buffer (Hanks, PBS) or serum-free basal medium; If subsequent operations such as fixation and perforation are required after staining, dilute the storage solution into mitochondrial assay working solution at a final concentration of 0.4-0.6 μM with biological buffer (Hanks, PBS) or serum-free basal medium;

2. Cell staining (the following protocol is for adherent cells; centrifugation is required for suspension cell exchange)

2.1. The cells are planted on cell culture plates or cell climbing plates until they converge to a certain extent;

2.2. The cell culture medium is removed and washed 1-2 times with preheated buffer for 3-5 min each time;

2.3. Add the preheated working solution for mitochondrial detection and incubate at 37°C for 30 min in a cell culture incubator (due to different cell types and states, the incubation time can be adjusted to some extent);

2.4. Remove the incubation solution and wash with buffer solution 2-3 times for 3-5 min each time;

2.5. Directly or with an Anti-fade Mounting Medium (recommended G1401) the films are sealed and observed under a fluorescence microscope (Ex=579 nm, Em=599 nm);

3. Cell fixation and perforation and other manipulations (optional):

Note: After staining, this product can be fixed and perforated, but the fluorescent signal will be attenuated after fixation.

3.1. Cell fixation

3.1.1. Cells treated with mitochondrial fluorescent dye labeling staining and washing are added to fixative (recommended G1101) and fixed for 10 min at room temperature;

3.1.2. Remove the fixative and wash with buffer 2-3 times for 3-5 min each time;

3.2. Cell perforation

3.2.1. The fixed and washed cells are added with 0.2-0.5% Triton X-100 and perforated for 10 min at room temperature;

3.2.2. The perforating fluid is removed and washed with buffer solution 2-3 times for 3-5 min each time;

3.3. The cells are stained and then fixed and perforated for processing, and can be used for the next experiments such as multilabeling.

Note

1. Due to the different types and states of the cells, the working concentration of the dye and the incubation time can be adjusted appropriately.

2. The dye needs to be used for mitochondrial localization in the state of living cells, and the cells cannot be fixed first, otherwise it will lead to false positive results of inaccurate localization. Fixation and subsequent operations can be performed after staining of living cells.

3. When staining and washing live cells, the assay working solution and washing buffer need to be pre-warmed at 37°C. Staining and washing are accomplished by incubation in an incubator so as not to affect the morphology of the cells by temperature difference stimulation.

4. In order to prevent fluorescence quenching, the entire staining process needs to be operated away from light.

5. Probe master mixes should be stored in separate containers to avoid repeated freezing and thawing.To avoid waste, the staining solution is prepared as needed, ready to use.

6. For your health and safety, please wear lab coat and gloves during operation.

For Research Use Only!

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