| Usage |
Self-brought instruments, reagents and consumables:
Cell culture plate, adjustable pipette gun and tip
Centrifuge
Fluorescence microscope or flow cytometry
PBS
Reagent Preparation:
Calcein AM staining solution: Add 1 μL of Calcein AM (1000X) per 1 mL of Assay buffer and mix well.
Note: The final concentration of Calcein AM needs to be optimized by pre-experiment according to different cell lines and experimental systems. The recommended working concentration of Calcein AM is 1 ×, which can be adjusted between 0.5 ×-5 ×.
Fluorescence quenching solution: 10 μL of CoCl per 1 mL of Calcein AM staining solution2(100 ×), mix well.
Note: CoCl2The final concentration is recommended to be 1 ×, and the quenching effect is usually better at this time. CoCl2The final concentration can also be appropriately optimized according to the type of cells used in the experiment to find the best quenching effect, and can be adjusted between 0.1 ×-1 ×.
Ionomycin control: Add 5 μL of Ionomycin (200 ×) per 1 mL of Fluorescence quenching solution and mix well.
Note: The final concentration of Ionomycin is recommended to be 1 × and can also be adjusted between 0.5 ×-5 ×.
Experimental procedure:
Note: This kit (100 T) 96-well plate can detect 1000 T when 100 μL of the detection system per well.
1. Flow cytometry detection:
1. Treat the cells in the expected method;
2. For non-adherent cells, centrifuge at 300 g for 5 min to collect the cells, wash them twice with PBS, and discard the PBS. For adherent cells, cells were first digested with trypsin (without EDTA), then centrifuged at 300 g for 5 min, centrifuged to collect cells, washed twice with PBS, and discarded PBS.
Note: Prepare cell samples suspended in Assay Buffer for negative control at flow cytometry.
3. Add appropriate volumes of Calcein AM staining solution, Fluorescence quenching solution, and Ionomycin control to resuspend cells, so that the cell density is about 1 × 106/mL, incubate at 37 ℃ in the dark for 30-45 min, and the optimal incubation time of different cells is different.
4. After incubation, centrifuge at 300g for 5 minutes to collect the cells. Add 1 mL of Assay Buffer to each sample, gently resuspend, and centrifuge at 300 g for 5 min to collect cells.
5. Cells were resuspended with 400 μL Assay Buffer and analyzed by flow cytometry.
2. Fluorescence microscope detection:
1. For adherent cells:
(1) Cultivate the cells on a suitable well plate in a CO2 cell incubator at 37 °C for at least 24 h, and then perform subsequent experiments.
(2) Treat the cells with the expected method, and incubate the cells without inducer to establish a negative control group.
(3) The cells were washed twice with PBS.
(4) Add appropriate volumes of Calcein AM staining solution, Fluorescence quenching solution, and Ionomycin control to the cells, usually 100 μL per well for 96-well plates, 250 μL per well for 24-well plates, 500 μL per well for 12-well plates, 1 mL per well for 6-well plates, and incubate at 37 ℃ in the dark for 30-45 minutes. The optimal incubation time of different cells is different.
(5) After incubation, replace it with fresh preheated culture medium at 37 ℃, and incubate at 37 ℃ for 30 minutes in the dark to ensure that the cellular esterase fully hydrolyzes Calcein AM to generate green fluorescent Calcein.
(6) Wash with PBS 2-3 times, and then add Assay Buffer to observe under a fluorescence microscope (Calcein AM is green fluorescence, Ex/Em = 494/517 nm).
2. For suspension cells:
(1) Cells were processed in the expected manner and counted.
(2) Take 300 g of appropriate cells and centrifuge them for 5 min, discard the supernatant, wash the cells twice with PBS, and discard the PBS.
(3) Resuspend cells with appropriate volumes of Calcein AM staining solution, Fluorescence quenching solution and Ionomycin control, respectively, so that the cell density is about 1 × 106/mL, incubate at 37 ℃ in the dark for 30-45 min, and the optimal incubation time of different cells is different.
(4) Centrifuge at 300g for 5 minutes, aspirate the supernatant, slowly add 1mL of preheated culture medium at 37 °C to resuspend the cells, and incubate at 37 °C for 30 minutes in the dark to ensure that the intracellular esterase fully hydrolyzes Calcein AM to generate green fluorescent Calcein.
(5) Centrifuge at 300g for 5 minutes, aspirate most of the culture medium, resuspend the cells and smear them, and observe under a fluorescence microscope.
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| Description |
mitochondrial permeablity transition pore (MPTP), also known as mitochondrial giant channel (magachannel), is a non-selective highly conductive channel existing between the inner and outer membranes of mitochondria, composed of a variety of protein complexes. Mitochondrial permeability transition pores (MPTP) may be involved in the release of mitochondrial components during cell death. The mitochondrial inner membrane of normal cells can maintain normal mitochondrial potential gradient to ensure cell respiration and energy supply. With Ca2+The intake and release of, a low-conductivity osmotic switching hole switches back and forth between opening and closing. When cells undergo apoptosis and pathological death, the mitochondrial membrane potential switching pore permeability is changed, and Ca2+Overload, oxidation of mitochondrial glutathione, increase of reactive oxygen levels, including subsequent release of cytochrome C, and decrease of mitochondrial membrane potential will all lead to the activation of mitochondrial permeability transition pores. Mitochondrial membrane permeability transition pore assay kit is a more direct detection method for mitochondrial permeability transition pore openness than only mitochondrial membrane potential analysis. The principle is: first, Calcein AM is loaded through passive transportation. The latter is a cell staining reagent that fluorescently labels living cells. It can easily penetrate the living cell membrane and be sheared by esterases in the cell to form a membrane-non-permeable polar molecule Calcein, which is retained in the cell, causing the cytoplasm, including mitochondria, to emit strong green fluorescence. Add CoCl2After that, the fluorescence from the cytoplasm was reduced by CoCl2Quenching, leaving only fluorescence within the mitochondria. As controls, cells can be loaded with Calcein AM and CoCl2At the same time, it was treated with Ionomycin to load the cells with more Ca2+, causing activation of mitochondrial permeability transition pores to quench mitochondrial fluorescence.
Product Components:
| Name |
50T |
100T |
Storage conditions |
| Calcein AM (1000 ×) |
50 μL |
100 μL |
-20 ℃, protected from light |
| CoCl2（100×） |
0.5 mL |
1mL |
-20℃ |
| Ionomycin (200 ×) |
250 μL |
500 μL |
-20 ℃, protected from light |
| Assay Buffer |
100mL |
100 mL × 2 |
-20℃ |
Product Features:
High safety: almost no toxicity to cells;
Compatibility with multiple detection methods: Can be applied to flow cytometers and fluorescence microscopes. |
| Instructions |
should bring along their own instruments, reagents, consumables: strong >
cell culture plate, adjustable moving fluid and spear gun
the centrifuge fluorescence microscope and flow cytometry instrument
PBS p >reagent preparation: strong > p >Calcein AM staining solution: each 1 mL Assay buffer with 1 & mu; L of Calcein AM (1000X) And blending.
note: Calcein AM final concentration should be based on different cell lines and the experimental system is optimized through an experiment. Calcein AM The recommended working concentration of calcein is 1× , can be at 0.5× -5× Make adjustments between.
Fluorescence quenching solution: 10 &mu per 1 mL Calcein AM staining solution; L CoCl2(100×) And blending.
note: CoCl 2 sub >recommended the final concentration of 1 & times; In general, the quenching effect is better at this time. The final concentration of CoCl2 can also be appropriately optimized according to the type of cells used in the experiment to find the best quenching effect, which can be in 0.1× -1× Make adjustments between.
Ionomycin control: Add 5&mu to every 1 mL Fluorescence quenching solution; L of Ionomycin (200×) , mix well.
Note: The recommended final concentration of Ionomycin is 1× , can also be used at 0.5× -5× Make adjustments between.
Experimental procedure:
Note: The detection system of this kit (100 T) 96-well plate is 100 μ It can detect 1000 T at L.
I. Flow cytometry:
1. The cells were treated as expected;
2, for non-adherent cells, cells were collected by centrifugation at 300 g for 5 min, washed twice with PBS, and the PBS was discarded. For adherent cells, cells were first digested with trypsin (without EDTA), then centrifuged at 300 g for 5 min, collected by centrifugation, washed twice with PBS, and discarded with PBS.
Note: A sample of cells suspended in Assay Buffer should be prepared to be used as a negative control during flow cytometry.
3. Add the appropriate volume of Calcein AM staining solution, Fluorescence quenching solution and Ionomycin control to resuspend the cells, respectively. The cell density was about 1× 10 6 sup >/ mL, 37 ℃ avoid light incubation 30-45 min, cells of different optimal incubation time is different.
4, After completion of incubation, cells were collected by centrifugation at 300g for 5 min. 1mL Assay Buffer was added to each sample, gently resuspended, and cells were collected by centrifugation at 300g for 5 min.
5, using 400 μ L Assay Buffer after heavy suspension cells, cells in the analysis. p >2, fluorescence microscope detection:
1, the adherent cells:
(1) on the suitable orifice plate culture cell, cell cultivation in 37 ℃ in CO2 at least 24 h, then follow-up experiments.
(2) Cells were treated with the expected methods and incubated without inducers to establish a negative control group.
(3) Cells were washed twice with PBS.
(4) Add appropriate volumes of Calcein AM staining solution, Fluorescence quenching solution, and Ionomycin control to the cells, respectively. Usually 100&mu was added to each well of 96-well plate; L, add 250 &mu to each well of a 24-well plate; L, add 500 &mu to each well of 12-well plate; 1 mL was added to each well of L, 6-well plates and incubated at 37 ° C in the dark for 30-45 min, with the optimal incubation time for different cells varying.
(5) At the end of the incubation, the cells were replaced with fresh culture medium preheated at 37℃ and incubated at 37℃ in the dark for another 30min to ensure that the intracellular esterase could fully hydrolyze Calcein AM to produce Calcein with green fluorescence.
(6) Wash with PBS for 2-3 times, then add Assay Buffer and observe under fluorescence microscope (Calcein AM is green fluorescence, Ex/Em=494/517nm).
2, for suspended cells:
(1) Cells were treated as expected and counted.
(2) Appropriate cells were centrifuged at 300 g for 5 min, the supernatant was discarded, and   was used; The cells were washed twice with PBS and the PBS was discarded.
(3) The cells were resuspended by adding appropriate volumes of Calcein AM staining solution, Fluorescence quenching solution, and Ionomycin control, respectively, so that the cell density was about 1× 106/mL, and the cells were incubated at 37 ° C in the dark for 30-45 min. The optimal incubation time was different for different cells.
(4) The cells were centrifuged at 300g for 5 min, the supernatant was removed, and the cells were resuspended by slowly adding 1mL of preheated culture medium at 37℃. The cells were incubated at 37℃ for another 30min in the dark to ensure that the intracellular esterase could fully hydrolyze Calcein AM to produce Calcein with green fluorescence.
(5) The cells were centrifuged at 300g for 5 min, most of the culture medium was removed by suction, and the cells were resuspended for smear and observed under a fluorescence microscope.
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