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    Luminescent Mycoplasma Detection Kit

    Luminescent Mycoplasma Detection Kit

    Catalog Number: abs90199 Brand: Absin
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    $70 USD
    $70 USD
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    Product Details

    Product Specification

    Usage 1. Bring your own consumables:
    Sterile centrifuge tube, adjustable pipette gun and gun tip
    centrifuge, 96 Well all-black or all-white ELISA plate
    sterilized water, PBS Or fresh medium
    Luminescence detector or multifunctional microplate reader
    2. Reagent preparation:
    Mycoplasma Assay Buffer ： Ready-to-use; Before use, equilibrate to room temperature; -20℃ Store away from light.
    Luciferase ： Ready-to-use; -20℃ Save.
    Mycoplasma Reagent A Preparation before use; use Mycoplasma Assay Buffer dissolve Mycoplasma Substrate A middle, Then the dissolved Mycoplasma Substrate A and Luciferase Join Mycoplasma Assay Buffer In the bottle, mix well to obtain... Mycoplasma Reagent A -20℃ ； Before use, equilibrate to room temperature.
    Mycoplasma Reagent B ： Ready-to-use; Before use, equilibrate to room temperature; Repackaging according to usage requirements -20℃ Save.
    Postive Control ： Ready-to-use; Before use, equilibrate to room temperature; Repackaging according to usage requirements -20℃ Save.
    Note: Avoid repeated freezing and thawing of all reagents. It is recommended to use them in separate packaging. The containers used in separate packaging must not ATP Pollution.
    III. Sample preparation:
    1 Adhesive cells: taken before cell digestion 0.2-1 mL Cell culture supernatant, 1,500 rpm centrifuge 5 min The supernatant was taken for detection.
    2 Suspension cells: Culture medium is taken during cell passage. 0.2-1 mL Cell culture medium, 1,500 rpm centrifuge 5 min The supernatant was taken for detection.
    3 Resuscitating cells: fresh complete culture medium is added to the cryopreserved cells after thawing, cultivate 1-2 h later retrieval 0.2-1 mL Cell culture medium, 1,500 rpm centrifuge 5 min The supernatant was taken for detection.
    Note:
    1 After cell passage or digestion, the mycoplasma detection signal will decrease. If samples are taken for testing after cell passage or digestion, they must be taken after passage or digestion is complete. 24 h Samples were taken later.
    2 The supernatant sample is best tested immediately after collection, or it can be tested in... 4℃ Place and test on the same day, or place in -80℃ Store for testing within six months. Cryopreserved samples must be equilibrated to room temperature before they can be used for testing.
    IV. Experimental Procedures
    1 ,in 96 Add them to the all-black or all-white enzyme label plates. 50 μL Samples to be tested, Postive Control Negative controls (such as sterilized water, PBS Fresh culture medium).
    2 Add to each well 50 μL Mycoplasma Reagent A Mix well and let stand at room temperature. 5 min Then, the chemiluminescence value was determined by a multifunctional microplate reader. RLUA 。
    3 Add to each well 50 μL Mycoplasma Reagent B Mix well and let stand at room temperature. 10 min Then, the chemiluminescence value was determined by a multifunctional microplate reader. RLUB 。
    4 Calculate the ratio ( Ratio ） =RLUB/RLUA 。 Reference Table 1 The ratio is greater than 1.2 This indicates mycoplasma contamination, and the ratio is less than 0.9 This indicates no mycoplasma contamination, and the ratio is... 0.9-1.2 The original sample can continue to be cultured during this period. 24-48 h The test was repeated later.
    Table 1. Analysis of results of mycoplasma detection kit by luminescence method
    Ratio Results Analysis treatment method
    ＜ 0.9 negative No processing required
    0.9-1.2 suspicious Continue isolation culture 24-48 h The test will be conducted later.
    ＞ 1.2 positive Discarded or isolated after cell sterilization treatment and treated with special mycoplasma prevention or removal reagents
    Note:
    1 The reading time per well of the multifunctional microplate reader is set as follows: 1,000 ms It is necessary to strictly follow the procedures Mycoplasma Reagent B 10 min Perform the test later, but do not do it earlier or later, otherwise it may affect the judgment of the results of samples with a ratio near the critical value.
    2 The optimal experimental temperature for this method is 20-25℃ Before use, ensure that the reagent naturally returns to room temperature. Do not heat the reagent in a water bath or in any other way. If the reagent needs to be stored for a long time, It is recommended to repackage according to the dosage for each experiment and avoid repeated freezing and thawing.
    3 The skin surface contains a large amount ATP Gloves must be worn when handling samples and conducting experiments to prevent contamination of samples and reagents that could lead to false negatives or false positives.
    4 Some samples may contain components that inhibit or enhance the reaction, resulting in false negatives or false positives, such as the addition of special drugs or the use of certain special culture solutions. In this case, the sample can be diluted. 10 The test is performed after doubling, and further determined based on the results obtained after dilution. Ratio To determine if there is mycoplasma contamination.
    5 Using different batches of kits and different instrument ratios for the same sample ( Ratio There may be differences, but this does not affect the qualitative judgment.
    V. Results Presentation:
                                    Table 2. Example of Detection Data of Luminescence Mycoplasma Detection Kit
    sample negative sample positive sample negative control positive control
    HEK293T Shangqing COS-7 Shangqing Hela Shangqing 10 Double dilution PBS DMEM Postive Control
    RLUA 250 241 274 391 302 344 307
    RLUB 80 118 10809 1279 90 109 26484
    Ratio 0.32 0.49 39.45 3.27 0.30 0.32 86.27
    Description         The luminescent mycoplasma detection kit utilizes the activity of mycoplasma-specific kinases and passes through ATP The detection relies on a luciferase-catalyzed luminescence reaction, which is performed by comparing the detection reagents before and after addition. ATP The change in content is used to determine whether the sample is contaminated with mycoplasma. After mycoplasma is cleaved, it releases specific kinases that interact with substrates and can catalyze... ADP Convert to ATP 。
            The entire experimental procedure consisted of two steps. The first step was to add mycoplasma detection reagents to the sample. A The original presence in the test sample ATP The content; The second step is to add a mycoplasma detection reagent. B If a sample is contaminated with mycoplasma, its unique kinase can catalyze... ADP Convert to ATP At this point, the original background is being tested. ATP Content and newly generated by mycoplasma-specific enzymes ATP The sum of the contents. By calculating the luminescence reading B with A The ratio can be used to determine whether there is mycoplasma contamination. If the ratio is greater than 1.2 This indicates mycoplasma contamination; a higher ratio indicates a higher degree of contamination. If the ratio is less than... 0.9 This indicates no mycoplasma contamination if the ratio is between... 0.9 and 1.2 In the meantime, it is recommended that the original cells (including the original culture medium) continue to be 24-48 h Test again later.
    Product Composition:
    components 20T 200T Preserve
    Mycoplasma Assay Buffer 1mL 10mL -20℃ Store away from light.
    Luciferase 1 μL 10 μL -20℃
    Mycoplasma Substrate A 1 1 -20℃ Store away from light.
    Mycoplasma Reagent B 1 mL 10 mL -20℃
    Postive Control 200 μL 1 mL -20℃
    Storage Temp. Store at -20℃ away from light and have a shelf life of 12 months.