| Product Description | Aladdin's UltraBio™ Plus Mycoplasma Elimination Reagent, abbreviated as UltraBio™ Plus, is a non-antibiotic reagent that can be used to prevent or eliminate mycoplasma in cell cultures rapidly and efficiently. It can completely 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 precious cells contaminated with mycoplasma or cells that have been contaminated with mycoplasma but need to be preserved.The usage of this product is basically the same as that of UltraBio™ Mycoplasma Elimination Reagent and Mynox® of Minerva Biolabs, with similar elimination efficiency, but this product is more convenient to use. Mycoplasma can be effectively eliminated by simply replacing the contaminated cell culture with new culture medium containing this product 1-3 times in 2-5 days, without any pretreatment needed.Mycoplasma is the smallest and simplest prokaryotic organism. As mycoplasma has no cell wall structures, antibiotics that target the cell wall, such as penicillin or β-lactam antibiotics, are ineffective against mycoplasma. Its size is approximately 0.2-0.8μm, therefore it can pass through 0.22μm filters and the conventional filtration method is ineffective to remove it either. Mycoplasma is usually adsorbed or scattered on cell surface and between cells to utilize the nutrition from hosts due to their limited ability for biosynthesis. Due to these characteristics, mycoplasma can severely impact contaminated cells in gene expression, metabolism, cell growth, differentiation, viability, and cell functions. Mycoplasma can seriously affect the reliability, reproducibility and consistency of experimental results and thus must be eliminated from cell cultures [1].This product can quickly and efficiently remove mycoplasma in cell cultures, with an elimination efficiency of nearly 100%. Most of the commercially available mycoplasma removal agents are tetracyclines, macrolides and quinolones antibiotics, which mainly inhibit mycoplasma but do not directly kill it, and thus have a slow and unsustainable effect [2-4]. The main component of this product is a peptide surfactant derived from the fermented extract of Bacillus subtilis. It selectively binds and alters the permeability of mycoplasma membrane, thus causing mycoplasma to rupture and die. Due to this property, the possibility of drug resistance is extremely low. After removing UltraBio™ Plus, cells can immediately return to their natural form and normal proliferation rate.Please refer to Figure 1 for the performance of this product in eliminating mycoplasma from contaminated adherent HeLa cells and suspension MOLM-13 cells.Figure 1. Removal of mycoplasma from adherent HeLa cells (A) and suspension MOLM-13 cells (B) with Aladdin's UltraBio™ Plus Mycoplasma Elimination Reagent . As shown in the figure, after eliminating mycoplasma twice with this product, cells can maintain mycoplasma-negative stably. Aladdin's Myco-Lumi™ Luminescent Mycoplasma Detection Kit for Low-sensitivity Instrument was used to determine whether there is mycoplasma contamination. A B/A ratio greater than 1.2 indicates the presence of mycoplasma and the larger the ratio, the higher the mycoplasma contamination. A B/A ratio less than 1.2 indicates no mycoplasma contamination, and the ratio between 0.9 and 1.2 indicates uncertainty. This figure is for reference only, which may vary due to different experimental conditions.This product works rapidly with stable efficacy. Mycoplasma in cell cultures can be eliminated almost permanently after 1-3 times treatment with this product in 2-5 days, which is more efficient and faster than using other mycoplasma removal reagents. Please refer to Figure 2 for the mycoplasma removal from adherent HeLa cells by using UltraBio™ Plus Mycoplasma Elimination Reagent , Mycoplasma Removal Agent , Mycoplasma Removal Agent Plus , Mycoplasma Prophylactic and Elimination Reagent I , and Mycoplasma Prophylactic and Elimination Reagen II . The antibiotic reagents require longer time of incubation and more times of action to remove or inhibit mycoplasma.Figure 2. Mycoplasma removal from adherent HeLa cells by using UltraBio™ Plus Mycoplasma Elimination Reagent , Mycoplasma Removal Agent , Mycoplasma Removal Agent Plus , Mycoplasma Prophylactic and Elimination Reagent I , and Mycoplasma Prophylactic and Elimination Reagen II . As shown in the figure, cells treated with UltraBio™ Plus maintained mycoplasma negative, while cells treated with other reagents returned positive during passaging. 1* and 2*, times of treatment; 1** and 2**, number of passages after cells were treated twice with each mycoplasma removal reagent. This figure is for reference only, which may vary due to different experimental conditions.This product containing no antibiotics will not cause drug resistance in mycoplasma and has low cytotoxicity. UltraBio™ Plus specifically kills mycoplasma by binding and altering the permeability of mycoplasma membrane. Although the viability and morphology of cells may be affected during the treatment, this product does not alter any cellular properties because it does not permeate the cell membrane of eukaryotic cells. After removing this product from cell culture, cells can quickly return to their normal growth and proliferation during subsequent passages.This product is a ready-to-use sterile solution. It can be added directly in new culture medium to replace the contaminated culture medium.C0285S, C0285M, and C0285L are sufficient for preparing 100, 500 and 2000ml of cell culture medium, respectively.
Precautions： Although Myco-Zero™ Plus 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.Myco-Zero™ Plus kills mycoplasma by binding to mycoplasma membrane directly. Therefore, for adherent cells, we recommend adding this product after cells are digested and passaged to ensure a full contact of cells with this product.Myco-Zero™ Plus may have a certain impact on the viability and the morphology of cells. To ensure the normal growth of cells after treatment, it is necessary to treat cells at a certain cell density, such as more than 60% for adherent cells, Taking a 6cm dish as an example, the number of suspension cells should be more than 300,000, but the number of cells should not be too large either. Otherwise, the mycoplasma elimination efficiency will be reduced. Different cells have different tolerance levels of this product, and the cell number can be adjusted appropriately according to the results. Cells should be closely observed during the treatment, and if there is cell deformation and slow growth, etc., replace a standard culture medium. The diluted Myco-Zero™ Plus Removal Reagent at 1:1500 or more can also be used, but the times of treatment and the incubation time may need to be increased.The time required for eliminating mycoplasma is closely related to the cell type, cell status, cell number, mycoplasma species, and severity of mycoplasma contamination, etc. After treatment with the Myco-Zero™ Plus, we recommend treating the cells with Myco-Zero™ Plus for a second time in order to completely remove mycoplasma and prevent the reoccurrence of contamination.Mycoplasma may still be detected false-positive in cells after treatment with Myco-Zero™ Plus. On one hand, mycoplasma membrane is lysed during the elimination process and the released mycoplasma DNA in culture medium may be detected. However, the extracellular DNA will be slowly degraded 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™ Plus is affected by many factors such as cell density and cell growth state. One time of treatment with this product 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 Myco-Lumi™ Luminescent Mycoplasma Detection Kit can avoid false positive results.Before eliminating mycoplasma from virus, host cells to be infected by virus should be assayed first to determine whether they are mycoplasma-free.The enveloped viruses are susceptible to be eliminated by the Myco-Zero™ Plus because their outlayer lipid membrane composition is similar to mycoplasma membrane,. 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. Prepare the Myco-Zero™ Plus working solutionPrepare an appropriate amount of Myco-Zero™ Plus working solution by mixing the Myco-Zero™ Plus Mycoplasma Elimination Reagent (1000X) and fresh cell culture medium in a ratio of 1:1000. For example, add 10μl of this product to 10ml of fresh standard culture medium (10% serum in culture medium is acceptable), and mix well before use. Note: The Myco-Zero™ Plus working solution needs to be prepared freshly. To remove mycoplasma from virus samples, use serum-free cell culture medium with different dilution ratios, as described below.2. Mycoplasma removal from adherent cellsa. Day 1: Digest adherent cells and count the number of cells. Seed the Myco-Zero™ Plus working solution with an appropriate amount of cells, then culture under normal conditions. Note: Cells should cover more than 60% of the bottom of the Petri dish or flask on the second day.b. Day 3: Change the culture medium with Myco-Zero™ Plus mycoplasma removal working solution if cells grow slowly and are not suitable for passage or passage again if cells reach the density of passage. If the morphology of the cells is affected, appropriately reduce the Myco-Zero™ Plus content in culture medium.Note: In general, two consecutive treatments can successfully remove mycoplasma, and the times of treatment and duration can be adjusted according to the severity of mycoplasma contamination and the status of cells.c. Examine the cell culture supernatant with Myco-Lumi™ Luminescent Mycoplasma Detection Kit ( Plus working solution. Count the cells, add an appropriate amount of Myco-Zero™ Plus working solution, then culture cells under normal conditions.b. Day 3: Replace with new Myco-Zero™ Plus working solution.Note: In general, two consecutive treatments can successfully remove mycoplasma, and the number of treatments and duration can be adjusted according to the severity of mycoplasma contamination and the status of the cells.c. Examine the cell culture supernatant with Myco-Lumi™ Luminescent Mycoplasma Detection Kit ( Plus working solution by mixing the Myco-Zero™ Plus Mycoplasma Elimination Reagent (1000X) and serum-free culture medium at a ratio of 1:800. For example, add 12.5μl of this product to 10ml of serum-free culture medium and mix well before use.b. Mix the virus solution and Myco-Zero™ Plus working solution at a ratio of 1:10. For example, add 100μl of virus solution to 1ml of Myco-Zero™ Plus working solution, and mix well by gentle vortex. Note: For better mycoplasma elimination effect, there should be less fetal bovine serum in virus solutions.c. Incubate at room temperature for 2h.d. Terminate the action of Myco-Zero™ Plus by adding the "Virus-Myco-Zero™ Plus Mixture" at 1:9. For example, add 1ml of "Virus-Myco-Zero™ Plus Mixture" to 9ml of cell culture, or directly add the “Virus-Myco-Zero™ Plus Mixture” to the cells to be infected for cell infection, as long as the final total volume is 10 times that of the "Virus-Myco-Zero™ Plus Mixture".Note: During vortex, make sure that the entire inner surface of the centrifuge tube has contact with the mixture.5. Mycoplasma removal from enveloped virusa. Prepare the Myco-Zero™ Plus working solution by mixing the Myco-Zero™ Plus Mycoplasma Elimination Reagent (1000X) and serum-free culture medium at a ratio of 1:4000. For example, add 2.5μl of this product to 10ml of serum-free culture medium and mix well before use.b. Mix and Myco-Zero™ Plus working solution at a ratio of 1:10. For example, add 100μl of virus solution to 1ml of Myco-Zero™ Plus working solution, and mix well by gentle vortex. Note: For better mycoplasma elimination effect, there should be less fetal bovine serum in virus solutions.c. Incubate at room temperature for 30min.d. Terminate the action of Myco-Zero™ Plus by adding the "Virus-Myco-Zero™ Plus Mixture" at 1:9. For example, add 1ml of "Virus-Myco-Zero™ Plus Mixture" to 9ml of cell culture, or directly add the “Virus-Myco-Zero™ Plus Mixture” to the cells to be infected for cell infection, as long as the final total volume is 10 times that of the "Virus-Myco-Zero™ Plus Mixture".Note 1: During vortex, make sure that the entire inner surface of the centrifuge tube has contact with the mixture.Note 2: This process can be performed multiple times for complete removal of mycoplasma.6. Mycoplasma removal from reagentsMycoplasma in reagents can be eliminated as described in " Mycoplasma removal from non-enveloped virus".
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