| Usage | I. Self-prepared materials:
Fluorescence microscope, low-speed centrifuge, EB staining solution, slides, coverslips
II. Operation steps (for reference only):
1. Staining of suspended cells:
(1) Dilute 10 μL of washing solution to 1 μL of washing solution with deionized water.
(2) Centrifuge at 800 g for 5 min, collect cells, wash cells once with 300-400 μL of 1 μL of washing solution, and discard the supernatant.
(3) Add an appropriate amount of 1 μL of washing solution to resuspend cells, count and adjust the cell concentration to 106/mL.
(4) Take 90 μL of cell suspension to a new EP tube, add 10 μL of MDC staining solution, and mix gently. Stain at room temperature in the dark for 15-30 minutes. (See Note 1)
(5) Centrifuge at 800g for 5 minutes, collect cells, wash cells twice with 300-400 μL of 1× washing solution, and discard the supernatant.
(6) Add 100 μL of buffered collection solution to resuspend cells, drop onto a slide and cover with a glass slide.
(7) Observe under a fluorescence microscope (blocking filter wavelength 512nm), count and photograph.
2. Staining of adherent cells:
(1) Dilute 10× washing solution to 1× washing solution with deionized water, remove the culture medium and wash cells with 1× washing solution.
(2) Dilute an appropriate amount of MDC staining solution 10-fold with 1× washing solution, then add to the well plate and incubate at room temperature in the dark for 15-30 minutes. (See Note 2)
(3) After staining, wash twice with 1× cleaning solution, 3 minutes each time.
(4) Observe under an inverted fluorescence microscope (blocking filter wavelength 512nm), count and take pictures.
III. Staining results:
Normal cells:
Cells are uniformly stained with yellow-green fluorescence
Apoptotic cells:
Chromatin is condensed, the cell nucleus is fragmented into dots, and is stained with dense and intense green particles of varying sizes
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| Description | Autophagy is the process by which cells, upon stimulation, engulf their own cytoplasm or organelles, ultimately degrading the engulfed material within lysosomes. Autophagosomes are double-membrane-bound, round or oval structures containing cytoplasm, long-lived proteins and abnormal protein aggregates, damaged or redundant organelles such as mitochondria, rough endoplasmic reticulum, and microbodies, as well as viruses and bacteria.
Monodansylcadaverine (MDC) is a fluorescent dye and an eosinophilic stain. It is commonly used as a specific marker for detecting autophagosome formation. Its excitation filter wavelength is 355 nm, and its blocking filter wavelength is 512 nm. The Cell Autophagy Staining Detection Kit (MDC Method) is suitable for autophagy staining in cultured cells. Also known as MDC Staining Solution, it can be used in combination with EB for double staining.
Product composition:
Component | Name | 100T | Save | Reagent A | MDC staining solution | 1mL | -20℃, avoid light | Reagent B | 10×Cleaning fluid | 20mL | 2-8℃ | Reagent C | Buffer collection solution | 10mL | 2-8℃ |
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| General Notes | 1. MDC staining solution and EB reagent can be used together to distinguish normal cells, apoptotic cells and necrotic cells. It is toxic to a certain extent, so please operate with caution. If combined staining is required, EB staining solution with a final concentration of 2 μM can be added to the cell suspension added with MDC staining solution, and then the normal operation can be carried out.
2. According to the principle of adding half the volume of culture medium to the well plate, a 10-fold diluted MDC staining solution can be added. The dilution ratio can also be adjusted as appropriate according to the number of cells to avoid false positives caused by excessive concentration.
3. During the operation, care should be taken to reduce the time the reagent is exposed to strong light.
4. For your safety and health, please wear a lab coat and disposable gloves when operating. |
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