Luminescent Mycoplasma Detection Kit

Cat. No.: L1506760

GRADE & PURITY BioReagent, for Cell culture, for Chemiluminescence, Sterile

Overview

  This luminescence-based mycoplasma detection kit utilizes the activity of mycoplasma-specific kinases and an ATP-dependent luciferase-catalyzed luminescence reaction. It determines mycoplasma contamination by comparing the change in ATP levels before and after adding the detection reagents. Upon lysis, mycoplasma release specific kinases that interact with substrates, catalyzing the conversion of ADP to ATP.

  The assay involves two steps. First, Mycoplasma Reagent A is added to the sample to measure the intrinsic ATP level. Second, Mycoplasma Reagent B is added. If mycoplasma contamination is present, their specific kinases catalyze the conversion of ADP to ATP. The luminescence measured in this step represents the sum of the intrinsic ATP and the newly generated ATP produced by the mycoplasma-specific enzymes. The presence of mycoplasma contamination is determined by calculating the ratio of Luminescence Reading B to Reading A. A ratio greater than 1.2 indicates mycoplasma contamination, with higher ratios suggesting greater contamination levels. A ratio less than 0.9 indicates no mycoplasma contamination. If the ratio falls 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 retest.

Applicable Sample: Cells

L1506760	Component	20T	200T	Storage
L1506760A	Mycoplasma Assay Buffer	1 mL	10 mL	-20℃. Store in the dark.
L1506760B	Luciferase	1 μL	10 μL	-20℃
L1506760C	Mycoplasma Substrate A	1 EA	1 EA	-20℃. Store in the dark.
L1506760D	Mycoplasma Reagent B	1 mL	10 mL	-20℃
L1506760E	Postive Control	200 μL	1 mL	-20℃

Materials Required but Not Provided

1. Sterile centrifuge tubes, adjustable pipettes and tips

2. Centrifuge, 96-well solid black or solid white microplate

3. Sterile water, PBS, or fresh culture medium

4. Luminometer or multifunctional microplate reader

Experimental Procedure

1. Reagent Preparation

Reagent Name	Preparation	Notes
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. Mix thoroughly to obtain Mycoplasma Reagent A.	Aliquot according to usage needs and store at -20°C protected from light. Equilibrate to room temperature before use.
Mycoplasma Reagent B	Ready-to-use; equilibrate to room temperature before use.	Aliquot according to usage needs; store at -20°C.
Postive Control	Ready-to-use; equilibrate to room temperature before use.	Aliquot according to usage needs; store at -20°C.

Note: Avoid repeated freeze-thaw cycles for all reagents. It is recommended to aliquot them. The aliquot containers must be free of ATP contamination.

2. Sample Preparation

2.1 Adherent Cells

Collect 0.2-1 mL of cell culture supernatant before trypsinization. Centrifuge at 1,500 rpm for 5 minutes. Collect the supernatant for detection.

2.2 Suspension Cells

During subculturing, collect 0.2-1 mL of the cell culture medium. Centrifuge at 1,500 rpm for 5 minutes. Collect the supernatant for detection.

2.3 Thawed Cells

After thawing frozen cells, add them to fresh complete medium. Culture for 1-2 hours, then collect 0.2-1 mL of the cell culture medium. Centrifuge at 1,500 rpm for 5 minutes. Collect the supernatant for detection.

Notes:

① The mycoplasma detection signal may decrease after cell passaging or trypsinization. If sampling after passaging or trypsinization, sample at least 24 hours after the process is complete.

② Supernatant samples are best detected immediately after collection. They can also be stored at 4°C and detected on the same day, or stored at -80°C for detection within six months. For detection, equilibrate frozen samples to room temperature before use.

3. Assay Steps

3.1 Add 50 μL of each test sample, Positive Control, and negative control (e.g., sterile water, PBS, fresh culture medium) into separate wells of a 96-well solid black or solid white microplate.

3.2 Add 50 μL of Mycoplasma Reagent A to each well. Mix well and incubate at room temperature for 5 minutes. Measure the chemiluminescence value (RLU_A) using a multifunctional microplate reader.

3.3 Add 50 μL of Mycoplasma Reagent B to each well. Mix well and incubate at room temperature for 10 minutes. Measure the chemiluminescence value (RLU_B) using a multifunctional microplate reader.

3.4 Calculate the Ratio = RLU_B / RLU_A. Refer to Table 1. A Ratio greater than 1.2 indicates mycoplasma contamination. A Ratio less than 0.9 indicates no mycoplasma contamination. If the Ratio is between 0.9 and 1.2, continue culturing the original sample for 24-48 hours and retest.

Table 1. Result Analysis for the Luminescence Mycoplasma Detection Kit

Ratio	Result	Recommended Action
＜0.9	Negative	No action required.
0.9-1.2	Suspect	Continue isolated culture for 24-48 hours and retest.
＞1.2	Positive	Discard cells after sterilization, or isolate and treat with specific mycoplasma prevention/removal reagents.

Notes:

① Set the reading time per well of the multifunctional microplate reader to 1,000 ms. Detection must be performed strictly 10 minutes after adding Mycoplasma Reagent B. Do not measure earlier or later, as this may affect the interpretation of results for samples with ratios near the cutoff value.

② The optimal temperature for this assay is 20-25°C. Ensure all reagents naturally equilibrate to room temperature before use. Do not heat reagents using a water bath or other methods. For long-term storage, aliquot reagents according to the amount needed per experiment and avoid repeated freeze-thaw cycles.

③ Skin surfaces contain significant ATP. Wear gloves when handling samples and performing the experiment to prevent contamination that could lead to false positives or negatives.

④ Some samples, such as those containing special additives or using certain culture media, might contain components that inhibit or enhance the reaction, potentially causing false negatives or positives. In such cases, dilute the sample 10-fold before detection and determine mycoplasma contamination based on the Ratio obtained from the diluted sample.

⑤ The Ratio for the same sample may vary when using different kit batches or different instruments, but this does not affect the qualitative determination.

4. Result Demonstration

Table 2. Example Detection Data for the Luminescence Mycoplasma Detection Kit

Frequently Asked Questions

Question	Answer
Are there sensitivity requirements for the luminometer used with this product?	A high-sensitivity instrument is recommended. If the ratio is consistently approximately 1, it might be related to the instrument's sensitivity. Try increasing the integration time from 1 second to an interval between 1-10 seconds.
How long can the prepared Mycoplasma Reagent A solution be stored?	It can be stored at -20°C protected from light for up to 3 months, or at -80°C protected from light for up to 12 months. Aliquoting is recommended to avoid repeated freeze-thaw cycles.
How does the luminescence method differ from PCR detection?	The chemiluminescence method is faster, allowing for convenient and high-throughput detection of mycoplasma contamination in cell culture supernatants.
Can the reaction time be extended to increase the signal for easier calculation?	No. The procedure must be followed strictly. Do not initiate reading earlier or later than specified, as this may affect the interpretation of results near the cutoff values.
Can the sample volume be changed to increase detection accuracy?	For a 96-well plate, 50 μL of sample is recommended. The sample volume can be increased (up to 100 μL maximum), with Mycoplasma Reagent A and B volumes increased proportionally. Reducing sample, Reagent A, or Reagent B volumes is not recommended, as detection values near the background may affect the ratio and result interpretation. If necessary, the sample can be diluted and tested.

Precautions

  This product is for research use only. Not for use in diagnostic procedures.

Storage and Shipping

Storage

Protected from light,Store at -20°C

Shipped In

Ice chest + Ice pads

Stability And Storage

Store at -20℃ long term (12 months). Store in the dark.