| Usage | 1. Preparation of working fluid:
1. Preparation of 1 × Assay Buffer
Take out 10 × Assay Buffer from low-temperature refrigerator, take out an appropriate amount according to the aseptic conditions of single dosage, and dilute 10 times with deionized water (dH2O) to obtain 1 × Assay Buffer.
2. Preparation of 1 × dyeing working solution
(1) First, return the cryopreserved Calcein-AM solution (2mM) and PI solution (1.5 mM) to room temperature for 20-30min.
Note: The mother liquor can be dispensed for the first use to reduce the number of repeated freezing and thawing.
(2) Add 5μl of Calcein-AM solution (2mM) and 15μl of PI solution (1.5 mM) to 5ml of 1 × Assay Buffer and mix well. At this time, the working solution concentration of Calcein-AM was 2 μM, and the working solution concentration of PI was 4.5 μM. Because the optimal staining conditions of different cell lines are different, it is recommended to do gradient experiments for the initial experiment to determine the optimal concentrations of Calcein-AM and PI. The principle of gradient screening is to use the lowest probe concentration to obtain the best fluorescence results.
Note: Due to the poor stability of Calcein-AM, this dyeing solution must be prepared immediately and used up the same day.
2. Dyeing steps:
1. For adherent cells, first digest the cells with a cell spatula or trypsin-EDTA, and then collect the cells by centrifugation (1000rpm, 3min). For suspended cells, cells were collected by direct centrifugation (1000 rpm, 3 min).
2. Remove the supernatant and fully wash the cells 2 to 3 times with 1 × Assay Buffer to fully remove the residual esterase activity.
3. Prepare the cell suspension with 1 × Assay Buffer to have a density of 1 × 105 ~ 1 × 106 cells/ml.
4. Take 100μl of staining working solution and add it to 200μl of cell suspension, mix well, and incubate at 37 °C for 15min.
Note: Incubation time can be extended to 30min if desired.
5. Use a 490 ± 10 nm excitation filter under a fluorescence microscope to simultaneously detect living cells (yellow-green fluorescence) and dead cells (red fluorescence). In addition, only dead cells were observed using a 545 nm emission filter. Detection can also be performed directly under a fluorescent microplate reader using a suitable filter.
Note: The following methods can be used to optimize the optimum working concentrations for the two fluorescent dyes.
(1) incubating the cells with 0.1% saponin or 0.1-0.5% digoxin for 10 minutes, or incubating the cells with 70% ethanol for 30 minutes, thereby preparing dead cells;
(2) Dead cells were stained with 0.1-10 μM PI solution to obtain the optimal working concentration for staining only the nucleus, but not the cytoplasm.
(3) Dead cell staining was performed with 0.1-10 μM Calcein-AM to obtain an optimal working concentration that would not stain the cytoplasm. Live cells are then stained at this concentration to see if the live cells can be stained |
| Synonym | Calcein-AM/PI |
| Description | Calcein-AM is a cell staining reagent that fluorescently labels living cells and fluoresces green (Ex = 490nm, Em = 515nm). Because it introduces acetylmethoxymethyl ester (AM) group on the basis of traditional Calcein, it increases hydrophobicity and makes it easily penetrate living cell membranes. Once in the cell, Calcein-AM (which itself does not fluoresce) is cleaved by esterases in the cell to form a membrane-impermeable polar molecule Calcein, which is retained in the cell and emits strong green fluorescence. Compared with other similar reagents (such as BCECF-AM and CFDA), Calcein, AM is the most suitable fluorescent probe for staining viable cells due to its extremely low cytotoxicity, and does not inhibit any cellular functions, such as proliferation and lymphocyte chemotaxis.
Because dead cells lack esterase, Calcein-AM is only used for cell viability testing and short-term labeling of living cells. Therefore, Calcein-AM is often used in combination with dead cell fluorescent probes such as propidium iodide (PI) to perform fluorescent double staining of living cells and dead cells at the same time. Propidium iodide (PI) cannot pass through the cell membrane of living cells, but can only pass through the disordered area of the dead cell membrane to reach the nucleus, and embedded into the DNA double helix of the cell to produce red fluorescence (Ex = 535 nm, Em = 617 nm), so PI only stains dead cells. Since both Calcein and PI-DNA can be excited at 490 nm, live and dead cells can be observed simultaneously with fluorescence microscope. With 545 nm excitation, only dead cells can be observed.
The working principle of this kit lies in the double dyes of Calcein-AM and PI to perform double staining labeling of living cells and dead cells, so as to analyze the level of living cells and dead cells. According to our optimized experimental system, a single staining of 200μl cell suspension can perform 500 tests.
Product Components:
| Components | Name | Specifications | Save | | A | Calcein-AM Solution (2mM) | 50 μL | Store dry at-20 ℃ protected from light | | B | PI Solution (1.5 mM) | 150 μL | Store dry at-20 ℃ protected from light | | C | 10 × Assay Buffer | 50mL | Store at-20 °C, and can be stored at 4 °C for regular use. |
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| General Notes | 1. Since Calcein-AM is very sensitive to humidity, if the required amount of Calcein-AM solution is taken every time, the lid must be tightly sealed. It is recommended to package and seal storage according to the single dosage. The Calcein-AM working fluid must be prepared for ready use.
2. Propidium iodide (PI) has certain carcinogenicity, so we must pay attention to protection during operation. If it comes into contact with the skin, it needs to be washed with tap water immediately.
3. For your safety and health, please wear a lab coat and disposable gloves. |
| Instructions | 1. Preparation of working solution:
1, 1× The preparation of Assay Buffer (reaction buffer)
Remove from the low temperature refrigerator for 10 times; Assay Buffer, according to the single dosage of sterile conditions to take out the appropriate amount, with deionized water (dH2O) to do 10 times dilution to get 1times; Assay Buffer.
2, 1× Dyeing the configuration of the working liquid
(1) the Calcein - AM solution to be kept at low temperature (2 mM) and PI solution (1.5 mM) back to the room temperature 20-30 min.
Note: The mother liquor can be divided for the first use to reduce the number of repeated freezing-thawing.
(2) apply 5 & mu; l Calcein-AM solution (2mM) and 15μ l PI solution (1.5mM) added to 5ml 1× Assay Buffer and mix well. At this time, the working solution concentration of Calcein-AM obtained is 2μ M, PI working liquid concentration is 4.5 & mu; M. As the optimal staining conditions differ for different cell lines, gradient experiments are recommended for initial experiments to determine the optimal concentration of Calcein-AM and PI. The principle of gradient screening is to use the lowest probe concentration to obtain the best fluorescence results.
Note: Due to the poor stability of Calcein-AM, this staining solution must be used on the go and used up the same day.
II. Staining steps:
1. For adherent cells, first digest the cells with a cell scraper or pancrease-EDTA, and then collect the cells by centrifugation (1000rpm, 3min). For suspended cells, cells were collected by centrifugation directly (1000 rpm, 3min).
2, remove the supernatant and use 1× The cells were thoroughly washed 2 to 3 times with Assay Buffer to fully remove residual esterase activity.
3, with 1× The cell suspension was prepared with Assay Buffer to a density of 1× 105~1× 106 cells /ml.
4, take 100μ l Add 200&mu to staining working solution; l cell suspension was mixed and incubated at 37 ° C for 15min.
Note: The incubation time can be extended to 30min if desired.
5, use 490± under fluorescence microscope; 10 nm excitation filter was used to detect both live cells (yellow-green fluorescence) and dead cells (red fluorescence). In addition, only dead cells could be observed using the 545nm emission filter. It can also be detected directly under a fluorescent microplate reader using a suitable filter.
Note: The following methods can be used to optimize the optimal working concentration of the two fluorescent dyes.
(1) Dead cells were prepared by incubating cells with 0.1% saponin or 0.1-0.5% digoxin for 10min or 70% ethanol for 30min;
(2) use 0.1-10μ M's PI solution was used for dead cell staining to obtain an optimal working concentration that stained only the nucleus, but not the cytoplasm.
(3) with 0.1-10μ M of Calcein-AM was used for dead cell staining to obtain an optimal working concentration that would not stain the cytoplasm. This concentration was then used for live-cell staining to see if viable cells could be stained |
| Storage Temp. | Stored dry at-20 ℃ in the dark from light, the shelf life is 12 months. |
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