1 / 12

Lab meeting 13.03.25

Lab meeting 13.03.25. Plasmid preparation and Linearize the pcDNA3.1 vector. Plasmid preparation using Midi kit from Quiagen. pcDNA3.1 digest with ScaI- blunt end . Set up protocol to determine Neomycin sensitivities of Hela .

ping
Download Presentation

Lab meeting 13.03.25

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Lab meeting 13.03.25

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

  3. Set up protocol to determine Neomycin sensitivities of Hela • 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 + 60μg/ml Neo + 440 μg/ml RPMI 10% FBS P.S • B5-6 : 500 μg/ml cells + 100μg/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 400 μg/ml 400 μg/ml 300 μg/ml Control Control 500 μg/ml 600 μg/ml 1000 μg/ml 1000 μg/ml 500 μg/ml 600 μg/ml 1200 μg/ml 1200 μg/ml

  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 and 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. Mycoplasma contamination & Effects on Cell Cultures • 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 presentation, 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

  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. Instruction for Testing • 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, blood • products from cell cultures) • Vaccines for humans and the veterinary field • Test necessary for: • Editors who are aware of the significance of mycoplasma contamination

  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

More Related