1. Lab meeting 13.03.25

2. Plasmid preparation and Linearize the pcDNA3.1 vector  pcDNA3.1 digest with ScaI- blunt end  Plasmid preparation using Midi kit from Quiagen

3. Set up protocol to determine Neomycin sensitivities of Hela 300 μg/ml 400 μg/ml 500 μg/ml 600 μg/ml 600 μg/ml 1000 μg/ml 1200 μg/ml Control  split a confluent plate so the cells will be approximately 25% confluent  Neomycin 10mg/ml  A1-2 : 500 μg/ml cells + 0 μg/ml Neo + 500 μg/ml RPMI 10% FBS P.S  A3-4 : 500 μg/ml cells + 30 μg/ml Neo + 470 μg/ml RPMI 10% FBS P.S  A5-6 : 500 μg/ml cells + 40 μg/ml Neo + 460 μg/ml RPMI 10% FBS P.S  B1-2 : 500 μg/ml cells + 50 μg/ml Neo + 450 μg/ml RPMI 10% FBS P.S  B3-4 : 500 μg/ml cells + 60 μg/ml Neo + 440 μg/ml RPMI 10% FBS P.S  B5-6 : 500 μg/ml cells + 100 μg/ml Neo + 400 μg/ml RPMI 10% FBS P.S  C1-2 : 500 μg/ml cells + 120 μg/ml Neo + 380 μg/ml RPMI 10% FBS P.S  Total volume of Hela cells + selective medium : 1 mL  Replenish the selective media every 2-3 days, and observe the percentage of surviving cells.  Count the number of viable cells at regular intervals to determine the appropriate concentration of antibiotic that prevents growth within 1–2 weeks after addition of the antibiotic. 300 μg/ml

4. 4 Contamination is a problem in cultures The problem and detection of contamination caused by aggressive cell lines, bacteria, mycoplasma, yeast, virus or fungus

5. What are Mycoplasma? Kind of bacteria Absence of cell wall, flexible membrane  easy taking in different shapes a nd difficulties in identifying under electron microscope Small size (0.15-0.3μm)  main reason for their escape through filtering systems and also their growth in high concentration in mammalian cell cultures without any turbidity or other obvious symptoms

6.  Inhibition of cell proliferation up to 50% by nutrient withdrawal and secretion of harmful metabolic products McGarrity et al. (1984) In Vitro Cell. Dev. Biol. 20:1 – fast glucose reduction and formation of acids => pH shift – arginine depletion => inhibition of protein biosynthesis, cell division and growth  Influence of immunological reactions (macrophage activation, inhibition of antigen prese ntation, induction of signal transduction) Muhlradt et al. (1996) Biochemistry 35:7781 – Influence of virus proliferation and the infection rate Nar-Paz et al.(1995) FEMS Microbiol. Lett. 128:63 – Cause chromosomal aberrations and multiple translocations McGarrity et al. (1978) In: Mycoplasma infection of cell cultures. – Disturbance of the hybridoma technique, contaminated cells become accumulation of mycoplasma at cells wall alters cell wall integrity Mycoplasma contamination & Effects on Cell Cultures

7. Effects on Cell Cultures (continued)  Significant changes in gene expression profiles  Mycoplasma can constitute up to 50% of the total protein and 15- 30% of the isolated DNA  Decrease of the transfection rates by 5% through electroporation  Induction of leopard cells (condensation of the chromatins)  Influence almost all functions of the host cell metabolism

8. Important of the Mycoplasma Tests 1) may show as abnormal cell growth, decreased growth rate, reduced saturation. 2) invisible under regular microscopy. 3) Standard antibiotics can allow contamination levels lower than detection levels, Pen/Strep does not provide protection from contamination 4) requires kits for testing.

9.  Regulations: FDA Points to Consider (May 1993), Regularien 21CFR610.30 USDA federal code #9CFR113.28 European Pharmacopoeia 2.6.7, Suppl. 5.8 ICH Guideline for biotechnological/biological products  Obliged to test: Master cell cultures, cell cultures, virus stocks, control cell cultures Bioproducts from cell cultures (antibodies, hormons, immune stimulators, b lood products from cell cultures) Vaccines for humans and the veterinary field  Test necessary for: Editors who are aware of the significance of mycoplasma contamination Instruction for Testing

10. Detection of Mycoplasma containmination on Hela DAPI staining

11. Detection of Mycoplasma containmination on Hela (continued) PCR method e-Myco™ plus Mycoplasma PCR Detection Kit Target band: 268 bp ~ 277bp Inetrnal control 160 bp Positive Negative Hela Ladder

12. Mycoplasma Elimination kit