This Mycoplasma detection kit is designed by using the activity of the unique enzyme in mycoplasma. The enzyme can decompose the specific substrate in the mycoplasma detection reagent and convert ADP into ATP. In the presence of ATP, the luciferase enzyme catalyzes the oxidation of luciferin to produce bioluminescence, which can be measured by luminometer. To reflect whether there is mycoplasma contamination in the samples to be tested. The whole detection process is simple and only needs two steps, which takes about 15 minutes. This method is highly sensitive and detects real biological active mycoplasma, so the detection result is more accurate than PCR method.

 

Wet ice transportation; Stored at -20℃, mycoplasma detection reagent A should be stored away from light. It is valid for 12 months.

 

composition

 

1. Take an appropriate amount of cell supernatant (1 mL is enough) for 3-6 days and centrifuge it at 400 g for 3 min to remove a small amount of floating cells or debris deposited. Take the supernatant for immediate detection, or store it at 4℃ for detection within 1 week, or store it at -80℃ for detection within half a year;

2. All test reagents and test samples are balanced to room temperature, the most suitable temperature is 20-25℃;

3. Add 50 μL sample and negative control (such as sterile water or PBS) to 96-well test plate (non-transparent plate, special 96-well white board is recommended);

4. Add 50 μL mycoplasma detection reagent A, mix gently to avoid bubbles, set aside at room temperature (20-25℃), dark for 5 min. Chemiluminescence detection was then carried out with A enzyme-plate reader with chemiluminescence detection, and the reading was A. (Please adjust the corresponding parameters according to the sensitivity of the instrument, the detection time of each hole is generally 0.25-1 s);

5. Add 50 μL of Mycoplasma detection reagent B, mix gently to avoid bubbles, and set aside at room temperature (20-25℃), away from light for 10 min. Chemiluminescence detection was then carried out with a enzyme-plate reader with chemiluminescence detection, and the reading was B. (Note: The detection should be carried out in strict accordance with 10 min after adding mycoplasma detection reagent B, and should not be carried out in advance or later, otherwise the judgment of results will be affected);

6. Calculate Ratio = Read value B/ Read value A.

A. If B/A>1.1, there is mycoplasma contamination in the cell culture;

B. If B/A<0.9, there is no mycoplasma contamination in the cell culture;

C. If the B/A ratio is between 0.9 and 1.1, it is recommended that cells be cultured for 24-48 h and tested again to determine whether there is mycoplasma contamination. If the B/A ratio is still between 0.9 and 1.1, the cell culture is free of mycoplasma contamination and mycoplasma negative.

1. Mycoplasma test reagent A contains luciferase. Repeated freezing and thawing will gradually deactivate it.

2. It is strongly recommended to use white or black opaque 96-well plate during detection, because the use of ordinary transparent 96-well plate will cause interference between adjacent detection holes.

3. The surface of human skin is rich in ATP, so please wear experimental gloves and masks when testing. Other consumables should also be clean and pollution-free to prevent the pollution caused by exogenous ATP.

4. For your safety and health, please wear lab coat and disposable gloves.

 

Products for scientific research purposes only, not for clinical diagnosis!