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Cat. No.: LMDKL-20 (for 20T)

Cat. No.: LMDKL-100 (for 100T)

Description

Luminescent Mycoplasma Detection Kit (for Low Sensitivity Instrument) is a kit that detects mycoplasma contamination in cultured cells and other biological materials through chemiluminescence.

The purpose of the Luminescent Mycoplasma Detection Kit (for Low Sensitivity Instrument) is the same as that of the Luminescent Mycoplasma Detection Kit (for High Sensitivity Instrument), with slight differences in performance. Please refer to the table below for specific details. Both products have been modified based on the sensitivity of the instruments used, making the detection kits more suitable for the corresponding detection instruments. Under the same detection instrument conditions, the readout value of the Luminescent Mycoplasma Detection Kit (for Low Sensitivity Instrument) is generally 10-50 times higher than that of the Luminescent Mycoplasma Detection Kit (for High Sensitivity Instrument). Therefore, the Luminescent Mycoplasma Detection Kit (for Low Sensitivity Instrument) is more suitable for low-sensitivity, relatively low-readout chemiluminescence instruments, such as Molecular Devices’ SpectraMax® M3. These instruments have lower readout values, and using the detection kit designed for low-sensitivity instruments ensures good chemiluminescence readings during detection. The Luminescent Mycoplasma Detection Kit (for Low Sensitivity Instrument) can also be used with high-sensitivity instruments, resulting in higher readout values but slightly lower detection sensitivity for mycoplasma, meaning the ratio of positive samples will be lower. On the other hand, the Luminescent Mycoplasma Detection Kit (for High Sensitivity Instrument) is more suitable for high-sensitivity, relatively high-readout chemiluminescence instruments, such as ThermoFisher’s multifunctional microplate readers Varioskan Flash/Varioskan LUX. These instruments have higher readout values, and using the detection kit designed for high-sensitivity instruments ensures higher detection sensitivity for mycoplasma. The Luminescent Mycoplasma Detection Kit (for High Sensitivity Instrument) is similar to Lonza’s MycoAlert™ Mycoplasma Detection Kit, while the Luminescent Mycoplasma Detection Kit (for Low Sensitivity Instrument) is similar to Lonza’s MycoAlert™ PLUS Mycoplasma Detection Kit. The detection sensitivity of the Luminescent Mycoplasma Detection Kit (for Low Sensitivity Instrument) is slightly lower than that of the Luminescent Mycoplasma Detection Kit (for High Sensitivity Instrument), specifically in that the ratio of positive samples will be lower, but it still meets the needs of routine detection. Please refer to the table below for a comparison and selection of the two kits.

Mycoplasma is the smallest and simplest prokaryote. Mycoplasma has the following characteristics:

• Mycoplasma lacks a cell wall structure, so many common antibiotics targeting the cell wall, such as penicillin or β-lactam antibiotics, are ineffective against it.
• Mycoplasma sizes range between bacteria and viruses, approximately 0.2-0.8μm, so some mycoplasma can pass through 0.22μm filters, making conventional filtration ineffective.
• Many mycoplasmas rely on the host for nutrients due to their limited biosynthetic capabilities, often adhering to or dispersing between cell surfaces.

These characteristics of mycoplasma pose a risk of contamination during cell culture, and mycoplasma contamination has become a global issue. Mycoplasma contamination can severely affect cell status, altering gene expression and metabolic characteristics, leading to slowed cell growth, abnormal differentiation, and death, significantly impacting cell function. These factors can seriously affect the reliability, reproducibility, and consistency of experimental results, making the detection of mycoplasma contamination very important.

Bacterial, yeast, or fungal contamination in cell culture can be seen under an optical microscope, but mycoplasma contamination is usually invisible under an optical microscope and must be detected using specific methods. Common methods for detecting mycoplasma contamination include mycoplasma isolation culture, ELISA, specialized biochemical detection, PCR detection, and DNA fluorescence staining detection. Most of these methods involve relatively cumbersome steps, low sensitivity, or long detection times.

The luminescent mycoplasma detection kit utilizes the activity of mycoplasma-specific enzymes and detects through an ATP-dependent luciferase-catalyzed luminescence reaction. It determines whether a sample is contaminated with mycoplasma by comparing the change in ATP levels before and after adding the detection reagent. The entire detection process involves only two steps and takes about 15 minutes. In the first step, mycoplasma detection reagent A is added to the sample, and after 5 minutes, the reading (A) is taken, which measures the original ATP level in the sample. In the second step, mycoplasma detection reagent B is added, and after 10 minutes, the reading (B) is taken. If the sample is contaminated with mycoplasma, its specific enzymes convert ADP in reagent B to ATP, and the reading measures the total ATP level, including the newly generated ATP catalyzed by mycoplasma-specific enzymes. By calculating the ratio of reading B to reading A, one can determine whether mycoplasma contamination is present. A ratio greater than 1.2 indicates mycoplasma contamination, with higher ratios indicating higher contamination levels. A ratio less than 0.9 indicates no mycoplasma contamination. If the ratio is between 0.9 and 1.2, it is recommended to continue culturing the original cells (including the original culture medium) for 24-48 hours and then test again.

This kit can quickly, effectively, and sensitively detect mycoplasma contamination. During cell culture, a small amount of culture supernatant can be taken at any time to monitor for mycoplasma contamination.

The luminescent signal of this kit is stable. After adding Mycoplasma Detection Reagent A, the luminescent signal stabilizes and can be detected in 5 minutes. After adding Mycoplasma Detection Reagent B, if the sample is free of mycoplasma contamination, the original luminescent signal will decrease to some extent. However, if the sample is contaminated with mycoplasma, the luminescent signal will increase over time and can be detected in 10 minutes.

If mycoplasma contamination is detected, it is recommended to replace the contaminated cells with uncontaminated ones for culture. If it is necessary to prevent or remove mycoplasma, our specialized mycoplasma prevention or removal reagents can be used.

This kit includes a certain amount of positive control, making detection more reliable and convenient. The positive control does not contain mycoplasma, eliminating the risk of mycoplasma contamination. It is recommended to set a positive control for each detection. The positive control can also be ordered separately (Luminescent Mycoplasma Detection Positive Control).

For 96-well plates, it is recommended to use 50μl of cell culture supernatant or other samples.

Components

• Mycoplasma Detection Reagent A
• Mycoplasma Detection Reagent B
• Positive Control

Storage

Store at -20°C for one year; store at -80°C for two years. Mycoplasma Detection Reagent A should be protected from light.

Precautions

• For first-time users of this kit, if you are unsure whether your instrument is low sensitivity or high sensitivity, it is recommended to first order the high sensitivity version. If the readout exceeds 10,000, it is likely a high sensitivity instrument.
• The Luminescent Mycoplasma Detection Kit (for Low Sensitivity Instrument) can also be used with high sensitivity instruments, resulting in higher readout values but slightly lower detection sensitivity for mycoplasma. However, the Luminescent Mycoplasma Detection Kit (for High Sensitivity Instrument) is not recommended for use with low sensitivity instruments, as the readout may be too low to accurately reflect mycoplasma contamination.
• Mycoplasma Detection Reagent A contains luciferase, which gradually loses activity with repeated freezing and thawing. Although testing has shown that up to 5 freeze-thaw cycles do not significantly affect detection performance, for optimal results, aliquot the reagent after the first thaw, ensuring the containers are free of ATP contamination. Repeated freeze-thaw cycles may cause slight precipitation in the detection reagent. In such cases, equilibrate to room temperature and dissolve as much as possible. If insoluble residues remain, centrifuge to remove them before use; this will not affect subsequent detection.
• Avoid repeated freezing and thawing of the positive control; aliquot and store as needed.
• It is strongly recommended to use white 96-well plates for detection. Using regular transparent 96-well plates may cause cross-well interference. White 96-well plates can be ordered from us.
• When detecting cells, culture them normally for at least 24 hours, preferably 3-6 days, and use the cell culture supernatant without trypsin digestion as the sample. For samples with extremely low mycoplasma content, such as serum, trypsin, antibiotics, and culture medium used for cell culture, it is best to culture for 2-3 days before testing. Shorter culture times may result in undetectable mycoplasma contamination. If only a short culture time is possible, consider using a mycoplasma PCR detection kit or other methods.
• For adherent cell culture supernatants containing a small number of cells or suspension cell cultures, centrifuge at 200×g for 5 minutes at room temperature and use the supernatant for detection to avoid high background values.
• Perform detection at room temperature (20-25°C) to ensure accurate results. Both the chemiluminescence reaction and the mycoplasma-specific enzyme-catalyzed reaction are temperature-sensitive. Equilibrate the detection reagents and samples to room temperature before detection.
• Human skin contains abundant ATP. Wear gloves during detection to prevent reagent contamination. Ensure other consumables are clean and uncontaminated.
• This product is for scientific research use only by professionals and is not intended for clinical diagnosis or treatment, food, or drugs, and should not be stored in ordinary residences.
• For your safety and health, wear a lab coat and disposable gloves when handling.

Only for research and not intended for treatment of humans or animals

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