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Q：What is Annexin V/PI assay?
The Annexin V/PI assay is used to distinguish early apoptotic cells through Annexin V labeling and necrotic/dead cells through propiconazole (PI) labeling. In the early stage of apoptosis, the cell membrane begins to lose asymmetry, leading to the translocation of phosphatidylserine (PS) to the outer surface of the membrane, where it can be detected by annexin V conjugates. However, in the case of membrane rupture, such as necrosis, annexin V conjugates can enter and bind to PS residues in the inner membrane, leading to false positive results. Therefore, it is recommended to use non membrane penetrating nuclear dyes (such as PI) in combination with annexin V conjugates. This allows users to distinguish between early apoptotic cells (Annexin V staining), late apoptotic cells (Annexin V and PI staining), and necrotic cells (PI staining only).
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Q：What is the difference between Annexin V and TUNEL?
Terminal deoxynucleotide transferase dUTP nick end labeling (TUNEL) is a staining method used to identify DNA fragmentation sites within cells - a hallmark feature of late stage cell apoptosis. It uses enzyme terminal deoxynucleotide transferase (TdT) to connect modified dNTPs (such as dUTP) to the 3 '- hydroxyl end of fragmented DNA strands. DNTPs are typically modified with fluorescent groups to facilitate quantification and/or visualization.
Annexin V staining identifies the early stages of cell apoptosis by binding to PS residues exposed outside the cell due to loss of membrane asymmetry. Annexin V is commonly labeled with fluorescent groups such as FITC to promote the detection of apoptotic cells.
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Q：Is the cell reactive oxygen species detection experiment applicable to all types of cells?
Cell reactive oxygen species detection experiments are typically applicable to most types of cells, including mammalian cells and microorganisms. However, different types of cells may have differences in the production and treatment of reactive oxygen species, so optimization and validation should be carried out for different types of cells when conducting experiments.
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Q：How to choose appropriate reactive oxygen species detection reagents?
When selecting reactive oxygen species detection reagents, factors such as cell type, experimental objectives, and detection methods should be considered. DCFH-DA is suitable for fluorescence microscopy observation, while ROS Glo reagent is suitable for flow cytometry analysis. Select appropriate reagents according to experimental requirements.
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Q：Can liquid culture media be stored at -20 ℃?
may not! Because at -20 ℃, salts in the culture medium are prone to precipitation. After the culture medium melts, some salts may not be able to dissolve again, resulting in a decrease in osmotic pressure of the culture medium. When cultivating cells, they are prone to rupture and death.
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Q：What is the difference between serum-free culture medium and regular culture medium?
Serum-free culture mediumIt is a synthetic culture medium that can maintain cell growth without the need for serum, and may contain small or large amounts of protein components.Ordinary culture mediumIt is usually necessary to add fetal bovine serum or other animal serum to promote cell growth.
The basic components of serum-free culture medium include basic culture medium and added components such as fibronectin, insulin, transferrin, etc., which are used to maintain the nutrients and factors required for cell growth.
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Q：How long can fetal bovine serum be stored at 4 ℃?
Fetal bovine serum can generally be stored for 6-12 months at 4 ℃. But this is not absolute, the actual storage time will be affected by various factors, including the quality of the serum, the sealing of the container, the stability of the refrigerator, etc. To ensure the quality of the serum, it is recommended to use it within 3 months after purchase. If long-term storage is required, it is best to freeze it at -20 ℃ or lower, which can last for 1-2 years or even longer. However, please note that repeated freezing and thawing should be avoided during each use to prevent loss of protein and nutrients. Before use, carefully read the product manual and follow the supplier's recommendations for operation.
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Q：What tests is the ROS detection kit suitable for?
It is generally used for the detection of mammalian cells and is only suitable for the detection of reactive oxygen species in live cells or living organisms.
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Q：Is the ROS detection kit suitable for detection in serum or plasma?
Not suitable for detecting ROS in serum or tissue homogenates. Fresh tissue prepared single-cell suspension can be attempted.
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Q：What are the reasons and solutions for FITC staining failure?
1. The cell clusters did not separate. Solution: It is recommended to set the centrifugal force at 500-1000g. If the centrifugal force is too low, only normal cells will be collected, and apoptotic cells and fragments will be lost, resulting in a low apoptosis rate; 2. No single staining control was conducted. Solution: The fluorescence value and background of each reagent kit are different, and the position of the cross door is also different. For example, compared to Li Ji, domestic American brands tend to lean to the left, while domestic brands tend to lean to the right. Therefore, it is recommended that customers do single dye door marking; 3. Too many cells. Solution: If there is no signal in the picture, it means that it was not detected, which means the signal exceeds the detection range. It is recommended to set the parameter to 10000 cells; 4. FITC cannot be stained. Solution: Generally, freezing will have an impact on FITC, and Li Ji's product recommendation is not to freeze it; 5. The concentration and duration of dye exposure, as well as incubation conditions, are also the main factors affecting staining. Solution: Different cells may not be exactly the same, and there are optimal conditions that need to be explored and confirmed. 6. Insufficient stimulation of cell apoptosis itself. Solution: Adjust the duration and concentration of drug action. 7. The state of the cell itself, such as cells that proliferate excessively and are insensitive to apoptotic stimuli. Solution: Confirm the cell state. 8. The digestion time of adherent cells is too long, the PS on the membrane surface is damaged, and the number of Annexin V binding sites decreases. Solution: Adjust the cell digestion time and avoid excessive digestion
"Heyuan Liji" is a joint venture between Heyuan Biotechnology (stock code: 688238) and Liji Biotechnology, specializing in the "Liji Biotechnology" and "Life-ilab" reagent brands
