BioMycoX® Mycoplasma Elimination Kit - QED Bioscience

BioMycoX® Mycoplasma Elimination KitQCS-E-01, QCS-E-03, QCS-E-05BioMycoX® Mycoplasma Elimination kit was developed for quick and complete elimination ofMycoplasma contamination in cell cultures. Commonly used methods for inactivation or elimination ofMycoplasma in cell cultures are antibiotic-based. These methods do not always result in successful orpermanent elimination of Mycoplasma. In addition, some antibiotics can have cytotoxic effects oneukaryotic cells and may lead to development of antibiotic-resistant strains of Mycoplasma. BioMycoX®Mycoplasma Elimination Kit is a non-antibiotic combination of biological reagents that completelyeliminate Mycoplasma contamination, often with just one treatment. And because it is not an antibiotic,development of resistant strains is highly unlikely.The active reagent in BioMycoX® Mycoplasma Elimination Kit integrates into the Mycoplasma membraneand compromises membrane integrity. The result is an osmotic influx that leads to completedisintegration of the Mycoplasma membrane. It does not cause any changes in normal eukaryotic cellcharacteristics. The BioMycoX® Mycoplasma Elimination Kit is suitable for the elimination of Mollicutesand related organisms (Mycoplasma, Acholeplasma, Ureaplasma, Spiroplasma, and Entomoplasma) incell cultures.Kit ContentsBioMycoX® Reagent 1: Vial with green capBioMycoX® Reagent 2: Vial with brown capTubes with filter capsStorage/StabilityThe kit is shipped at ambient temperature. Upon receipt, store at 4-8 o C. The reagents are stable until theexpiration date shown on the product label.Instructions for UseBioMycoX® Reagent 1 treatment must be conducted in media containing a maximum of 5% (v/v)fetal bovine serum (FBS). It is not necessary to remove antibiotics from media.A. Treatment of adherent cells:1. Trypsinize cells (80-90% confluent) by usual method. Check for complete detachment of cells.2. Centrifuge cell suspension at 1,500 rpm for 5 minutes. Decant supernatant.3. Resuspend cell pellet in 10ml of culture medium containing 5% FBS.4. Filter any clumped cells out of the cell suspension with a tube with filter cap included in the kit.If cell suspension does not readily pass through the filter, gently pipette up and down.5. Resuspend cells to a density of 10 6 cells/ml in culture medium containing 5% FBS.6. Add 3ml of the cell suspension and 200ul of BioMycoX® Reagent 1 to a sterile 15ml conicaltube.7. Adjust the volume of the cell- BioMycoX® Reagent 1 mixture to 10ml with culture mediumcontaining 5% FBS. Mix carefully.8. Transfer the 10ml from step 7 to a sterile 100mm culture dish or T75 culture flask and incubate ascells are normally cultured for 3 days.9. Trypsinize cells by usual method. Check for complete detachment of cells.10. Centrifuge the cell suspension at 1,500 rpm for 5 minutes. Decant supernatant.11. Resuspend cell pellet in 5 ml of culture medium containing 10% FBS.12. Filter any clumped cells out of the cell suspension with a tube with filter cap included in the kit.If cell suspension does not readily pass through the filter, gently pipette up and down.

BioMycoX® Mycoplasma Elimination Kit (continued)13. Add 4ml of the filtered cell suspension and 200ul of BioMycoX® Reagent 2 to a sterile 15mlconical tube.14. Adjust the volume of the cell-BioMycoX® Reagent 2 mixture to 10ml with culture mediumcontaining 10% FBS. Mix carefully.15. Transfer the 10ml from step 14 to a sterile 100mm culture dish or T75 culture flask and incubateas cells are normally cultured for 2-3 days.16. Repeat steps 9-15 two more times.17. Confirm the success of the treatment by testing with BioMycoX® Mycoplasma PCR Detection Kit.B. Treatment of suspension cells:1. Prepare cell suspension as normally done.2. Filter any clumped cells out of the cell suspension with a tube with filter cap included in the kit.If cell suspension does not readily pass through the filter, gently pipette up and down.3. Adjust the cell suspension to a cell density of 10 6 cells/ml in culture medium containing 5% FBS.4. Add 3ml of cell suspension and 200ul of BioMycoX Reagent 1 to a sterile 15ml conical tube.5. Adjust the volume of the cell-BioMycoX Reagent 1 mixture to 10ml with culture mediumcontaining 5% FBS. Mix carefully.6. Transfer the 10ml from step 5 to a sterile 100mm culture dish or T75 culture flask and incubate ascells are normally cultured for 2-3 days.7. Prepare the cell suspension again as normally done.8. Filter any clumped cells out of the cell suspension with a tube with filter cap included in the kit.If cell suspension does not readily pass through the filter, gently pipette up and down.9. Adjust the cell suspension to a cell density of 10 6 cells/ml in culture medium containing 10% FBS.10. Add 4ml of cell suspension and 200ul of BioMycoX Reagent 2 to a sterile 15ml conical tube.11. Adjust the volume of the cell-BioMycoX Reagent 2 mixture to 10ml with culture mediumcontaining 10% FBS. Mix carefully.12. Transfer the 10ml from step 11 to a sterile 100mm culture dish or T75 culture flask and incubateas cells are normally cultured for 2-3 days.13. Repeat steps 7-12 two more times.14. Confirm the success of the treatment by testing with BioMycoX Mycoplasma PCR Detection Kit.ResultsM: 100bp DNA ladder1: Positive control (Mycoplasma controlDNA)2: Negative control3 and 6: Mycoplasma-infected cells beforeBioMycoX® treatment4 and 7: Mycoplasma-infected cells / 1 passageafter BioMycoX® treatment5 and 8: Mycoplasma-infected cells / 10passages after BioMycoX® treatment

BioMycoX® Mycoplasma Elimination Kit (continued)BioMycoX® Mycoplasma Elimination Kit is manufactured for QED Bioscience bywww.cells-safe.com