| Product Description | Aladdin's UltraBio™ Mycoplasma Elimination Reagent, abbreviated as UltraBio™, is a non-antibiotic reagent for the rapid and efficient removal of mycoplasma from cell cultures. It can completely remove and eliminate mycoplasma without the need of long-term maintenance, and is suitable for the treatment of all kinds of mycoplasma-contaminated cells, viruses and related reagents, especially for precious cells contaminated with mycoplasma or cells that have been contaminated with mycoplasma but need to be preserved. Mycoplasma is the smallest and simplest prokaryotic organism. Mycoplasmas have the following characteristics
Precautions： Although Myco-Zero™ can completely eliminate mycoplasma, recontamination may still occur due to new contaminations from new cells, reagents, and surrounding environments. We recommended performing mycoplasma detection once a week during cell culture or as soon as possible after recovery of frozen cells. Cell density is an important factor affecting the efficacy of Myco-Zero™ in eliminating mycoplasma. For 6cm culture dishes, cell numbers must be strictly controlled between 1 and 100,000. Before using Myco-Zero™ for mycoplasma elimination, it must be ensured that cells are not in clumps. Myco-Zero™ kills mycoplasma by binding to mycoplasma membrane and therefore must come into direct contact with mycoplasma to be effective. Cell clumping tends to create cellular gaps and become a hiding place for mycoplasma, thus affecting the efficacy of Myco-Zero™. Cell clumping can be avoided by extending the duration time of trypsin digestion. The concentration of fat and protein in culture medium can affect the efficacy of Myco-Zero™ in eliminating mycoplasma. Fetal bovine serum contains cholesterol and other Myco-Zero™ target molecules that may prevent Myco-Zero™ from binding to mycoplasma membrane, and thus high concentrations of fetal bovine serum can severely affect the efficacy of Myco-Zero™ in eliminating mycoplasma. When eliminating mycoplasma from cell cultures with this product, the optimal concentration of fetal bovine serum in cell cultures is 5%. Cells should be added to the mixture of Myco-Zero™ and culture medium or PBS to enable full suspension of cells without the formation of aerosols. Aerosols adhering to the surface of the culture dish or centrifuge tube prevent the full contact between Myco-Zero™ and cells, thus affecting the elimination of mycoplasma. When treating cells with the Myco-Zero™ for several days (time to proliferate for one generation), cell growth state should be closely noted during this period. If necessary, the culture medium containing Myco-Zero™ can be replaced by standard culture medium or diluted 5 times with standard culture medium. Since Myco-Zero™ has certain cytotoxicity during the treatment, a control without Myco-Zero™ treatment can be set up at the same time, and passage when control cells are fully confluent. Mycoplasma may still be detected in cells after Myco-Zero™ elimination. On the one hand, mycoplasma membrane is lysed during the elimination process and mycoplasma DNA is released in the culture medium, causing false positive results. However, the extracellular DNA will be slowly hydrolyzed during cell growth, therefore, it is recommended to perform mycoplasma assay at 3-4 generations after mycoplasma elimination. On the other hand, the removal efficacy of Myco-Zero™ is affected by many factors such as cell density and cell growth state. The first treatment may not completely eliminate mycoplasma and may even cause recontamination of cells due to improper handling. A second treatment can be performed after the cell growth state is stabilized. Using ’s Myco-lumiTM Luminescent Mycoplasma Detection Kit can avoid false positives of nucleic acid assays. Before eliminating mycoplasma from virus, host cells to be infected by virus should be assayed first to ensure mycoplasma-free host cells are used. The enveloped viruses are susceptible to be eliminated by the Myco-Zero™, because their outlayer lipid membrane composition, similar to mycoplasma membrane, is also the target of Myco-Zero™. For complete removal of mycoplasma from virus while maintaining viral infectivity, the starting viral titer should be higher than 106 TCID50 (50% Tissue Culture Infectious Dose). To remove mycoplasma from non-enveloped viruses, the elimination efficacy of this product is not affected by viral titers. This product is for R&D only. Not for drug, household, or other uses. For your safety and health, please wear a lab coat and disposable gloves during the operation.
Instructions for Use： 1.Mycoplasma removal from adherent cellsa.Preparation of mycoplasma-contaminated cells: Digest and count the adherent cells, then adjust the cell density to 5000-50,000 cells/ml with the standard culture medium containing 5% fetal bovine serum. b.Add 2.8ml of standard culture medium containing 5% fetal bovine serum to a 6cm petri dish.c.Add 200µl of Myco-Zero™ and mix well.d.Add 2 ml of cell suspension prepared in step 1a and mix well. At this point the total volume is 5ml with 10,000-100,000 cells.e.Culture the cells normally until 80-90% confluence (usually 5-8 days) and perform cell passaging.Note: The cell growth state needs to be closely observed during the culture period. When cells are found to have obvious damages, stop the mycoplasma removal process by replacing or diluting the culture medium. If the cell growth is slow and the cell density does not reach 80-90% confluence after 5-8 days of culture, the cells can be transferred to a new culture dish after cell dissociation and the addition of standard culture medium.2.Mycoplasma removal from suspension cellsa.Preparation of mycoplasma-contaminated suspension cells: Count the cells and adjust the cell density to 6000-60,000 cells/ml with the standard culture medium containing 10% fetal bovine serum.b.Add 1.6ml of PBS containing 0.125% trypsin and 5mM EDTA into a 15ml sterile centrifuge tube. It is possible to use and mix well.d.Add 1.6ml of cell suspension prepared in step 2a. The total volume at this point is 3.4ml with with 10,000-100,000 cells.e.Incubate at 37℃ with slow shaking on a shaker for 30 minutes. Note: For Myco-Zero™ sensitive cells, the Myco-Zero™ action time can be adjusted to 15 to 20 minutes.f.Centrifuge at 600×g for 10 minutes and aspirate the supernatant.g.Add 5ml of standard culture medium containing 10% fetal bovine serum to resuspend cells, and transfer to culture dishes or culture flasks for normal culture.(Optional) For a thorough elimination of mycoplasma, proceed to the following steps:h.Add 150µl of Myco-Zero™ and mix well.i.Culture normally for 3 days.j.Replace with new standard culture medium containing 10% fetal bovine serum and continue normal culture.3.Mycoplasma removal from non-enveloped virusa.Add 1ml of serum-free standard culture medium to a 1.5ml sterile centrifuge tube.b.Add 100µl of Myco-Zero™ and mix well.c.Add 125µl of virus solution (with ≤8% fetal bovine serum). Vortex gently to mix well.Note: During the vortex, make sure that the entire inner surface of the centrifuge tube is sufficiently moistened with the mixture.d.Incubate at room temperature for 2 hours.e.Terminate the action of Myco-Zero™ by diluting the Virus-Myco-Zero™ mix with new cell culture medium at a ratio of 1:9 (e.g., adding 10.8ml of cell culture medium to 1.2 ml of Virus-Myco-Zero™ mix). The Virus-Myco-Zero™ mix can also be added directly to cells to be infected, as long as the final total culture volume is 10 times that of the Virus-Myco-Zero™ mixture. 4.Mycoplasma removal from enveloped virusa.Add 4.4ml of serum-free standard medium to a 15ml sterile centrifuge tube.b.Add 100µl of Myco-Zero™ and mix well.c.Add 500µl of virus solution (with ≤8% fetal bovine serum). Vortex gently to mix well.Note: During the vortex, make sure that the entire inner surface of the centrifuge tube is sufficiently moistened with the mixture.d.Incubate at room temperature for 30 minutes.e.Terminate the action of Myco-Zero™ by diluting the Virus-Myco-Zero™ mix with new cell culture medium at a ratio of 1:9 (e.g., adding 9ml of cell culture medium to 1ml of Virus-Myco-Zero™ mix). The Virus-Myco-Zero™ mix can also be added directly to cells to be infected, as long as the final total culture volume is 10 times that of the Virus-Myco-Zero™ mix.Note 2: This process of mycoplasma removal from virus solutions can be applied several times to ensure complete removal.5.Mycoplasma removal from reagentsMycoplasma in reagents can be eliminated by the protocol as described in " Mycoplasma removal from non-enveloped virus".References:1.Uphoff CC, Drexler HG. Curr Protoc Mol Biol. 2014. 106:28.4.1-14. 2.Fleckenstein E, Uphoff CC, Drexler HG. Leukemia. 1994. 8(8):1424-1434. 3.Uphoff CC, Gignac SM, Drexler HG. J Immunol Methods. 1992. 149(1):55-62.4.Blanchard A, Montagnier L. Annu Rev Microbiol. 1994.48:687-712.Related Products: Cat. No. Product Name Pack Size C0280S Myco-Zero™ Mycoplasma Elimination Reagent 5T C0280M Myco-Zero™ Mycoplasma Elimination Reagent 20T C0280L Myco-Zero™ Mycoplasma Elimination Reagent 100T C0283-500ml Myco-Zero™ Mycoplasma Elimination Spray 500ml C0283-2L Myco-Zero™ Mycoplasma Elimination Spray 2L C0288S Mycoplasma Removal Agent 20mg C0288M Mycoplasma Removal Agent 100mg C0290S Mycoplasma Removal Agent Plus 10mg C0290M Mycoplasma Removal Agent Plus 50mg C0292-2ml Mycoplasma Prophylactic and Elimination Reagent I 2ml C0292-10ml Mycoplasma Prophylactic and Elimination Reagent I 10ml C0293-2ml Mycoplasma Prophylactic and Elimination Reagent II 2ml C0293-10ml Mycoplasma Prophylactic and Elimination Reagent II 10ml C0296 Mycoplasma Stain Kit ＞100T C0297S Myco-Lumi™ Luminescent Mycoplasma Detection Kit(for Low-Sensitivity Instrument) 20T C0297M Myco-Lumi™ Luminescent Mycoplasma Detection Kit(for Low-Sensitivity Instrument) 100T C0298S Myco-Lumi™ Luminescent Mycoplasma Detection Kit (for High-Sensitivity Instrument) 20T C0298M Myco-Lumi™ Luminescent Mycoplasma Detection Kit (for High-Sensitivity Instrument) 100T C0299S Myco-Lumi™ Luminescent Mycoplasma Detection Positive Control 20T C0301S PCR Mycoplasma Detection Kit 250T
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