Luminescent Mycoplasma Detection Kit

1. Supplies to be provided: Sterile centrifuge tubes, adjustable pipettes and tips, Centrifuge, 96-well all-black or all-white ELISA plates, Sterile water, PBS, or fresh culture medium, Luminescence analyzer or multi-function microplate reader.

2. Reagent Preparation:

Mycoplasma Assay Buffer: Ready-to-use; equilibrate to room temperature before use; store at -20°C, protected from light.

Luciferase: Ready-to-use; store at -20°C.

Mycoplasma Reagent A: Prepare immediately before use; dissolve Mycoplasma Substrate A in Mycoplasma Assay Buffer. Then, add the dissolved Mycoplasma Substrate A and Luciferase to the Mycoplasma Assay Buffer bottle and mix thoroughly to obtain Mycoplasma Reagent A. Aliquot as needed and store at -20°C in the dark. Equilibrate to room temperature before use.

Mycoplasma Reagent B: Ready-to-use; equilibrate to room temperature before use; aliquot as needed and store at -20°C. 

Postive Control: Ready-to-use; equilibrate to room temperature before use; aliquot as needed and store at -20°C. 
Note: Avoid repeated freeze-thaw cycles for all reagents. Aliquoting is recommended. The aliquot containers must be free of ATP contamination.

III. Sample Preparation:
1. Adherent Cells: Before cell digestion, remove 0.2-1 mL of cell culture supernatant and centrifuge at 1,500 rpm for 5 minutes. The supernatant is then collected for testing.
2. Suspension Cells: When passaging cells, remove 0.2-1 mL of cell culture medium and centrifuge at 1,500 rpm for 5 minutes. The supernatant is then collected for testing.
3. Thawed Cells: After thawing, add fresh complete medium to frozen cells. After culturing for 1-2 hours, remove 0.2-1 mL of cell culture medium and centrifuge at 1,500 rpm for 5 minutes. The supernatant is then collected for testing.
Note:
1. Mycoplasma detection signal will decrease after cell passaging or digestion. If sampling for testing after cell passaging or digestion, it must be done 24 hours after completion of passaging or digestion.
2. Supernatant samples are best tested immediately after collection. Alternatively, they can be stored at 4°C and tested the same day, or stored at -80°C and tested within six months. Samples stored at low temperatures must be equilibrated to room temperature before testing.

IV. Experimental Procedure:
1. Add 50 μL of the sample to be tested, a positive control, and a negative control (e.g., sterile water, PBS, or fresh culture medium) to a 96-well all-black or all-white microtiter plate.
2. Add 50 μL of Mycoplasma Reagent A to each well, mix, and let stand at room temperature for 5 minutes. Determine the chemiluminescence value (RLUA) using a multi-functional microplate reader.
3. Add 50 μL of Mycoplasma Reagent B to each well, mix, and let stand at room temperature for 10 minutes. Determine the chemiluminescence value (RLUB) using a multi-functional microplate reader.
4. Calculate the ratio (Ratio) = RLUB/RLUA. Refer to Table 1. A ratio greater than 1.2 indicates mycoplasma contamination, a ratio less than 0.9 indicates no mycoplasma contamination, and a ratio between 0.9 and 1.2 can be used to culture the original sample for 24-48 hours before retesting.

Note:
1. The reading time for each well of the multifunctional microplate reader should be set to 1,000 ms. The assay should be performed strictly 10 minutes after the addition of Mycoplasma Reagent B. Do not read the assay earlier or later than this time, as this may affect the results of samples with ratios near the critical value.

2. The optimal experimental temperature for this method is 20-25°C. Ensure that the reagents have returned to room temperature naturally before use. Do not heat the reagents in a water bath or other means. For long-term storage, it is recommended to aliquot the reagents according to the amount used for each experiment and avoid repeated freeze-thaw cycles. 

3. The skin surface contains a large amount of ATP. Gloves should be worn when handling samples and conducting experiments to prevent contamination of samples and reagents, which could result in false negatives or false positives.

4. Some samples, if treated with special medications or culture media, may contain components that inhibit or enhance the reaction, leading to false negatives or false positives. In such cases, the sample can be diluted 10-fold before testing, and the ratio determined after dilution can be used to determine whether there is mycoplasma contamination.

5. The ratio value may vary when using different batches of test kits or different instruments for the same sample, but this does not affect the qualitative judgment.