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What aspects of cell culture media can be customized? Product FAQ

Answer

The Gibco Media Configurator allows you to add, remove, and adjust the concentration of components. In addition, you can select from a range of packaging, QC tests, and either cGMP or non-cGMP manufacturing.

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What factors must be considered when selecting a crosslinker? Product FAQ

Answer

 

When selecting a crosslinker, it is important to consider the following factors:


  • In vivo vs in vitro labeling: If labeling live cells, the crosslinker must be cell permeable unless you are crosslink proteins only on the membrane surface. If requiring a crosslinker that does not diffuse through the plasma membrane, use a branched chain crosslinker.
  • Metabolic vs random labeling: We offer modified amino acids and other substrates that can be added to culture medium and incorporated into proteins or other biomolecules during protein synthesis, post-translational modification, replication or other synthetic pathways. 
  • Reactive groups on the target: Do you know what reactive groups are available or are sites of interest on the proteins or other targets? Do you require a bifunctional crosslinker or trifunctional crosslinker? We offer crosslinkers that can react with amines, azides, carboxyls, carbonyls, hydroxyls and sulfhydryls in both homo- and heterobifunctional forms and trifunctional crosslinkers. When the reactive group on the target is unknown and linkage may be random, consider using a non-specific photoreactive crosslinker. The photoreactive crosslinkers can bind to primary amines, double bonds or insert into C-H and N-H sites. 
  • Timing of the linkage: Spontaneous crosslinkers will bind upon contact with the appropriate reactive group on the protein or other molecules. The rate of reaction depends on the reaction conditions, the concentration of the target and crosslinker, and the rate of diffusion. If a target is expressed or active during a distinct cellular event or under the control of an inducer and you wish to label the protein at this specific event or induction, the photoreactive crosslinkers can be activated upon exposing the sample to light at the desired time and duration. Various modified amino acids and other biomolecules can be incorporated by live cells by adding the substrate to the culture media. You would want to control the timing of the addition of the substrate to the cells to coincide with a cellular event or treatment. 
  • Solubility: Most in vitro crosslinking reactions are performed under mild physiological conditions. In some instances when a molecule is insoluble in aqueous buffers, some crosslinking reactions can be done in organic solvents such as acetonitrile, DMSO or DMF.
  • Distance between molecules: We offer zero-length crosslinkers as well as crosslinkers with spacer arms of various lengths, from zero length to >100 angstroms. Longer spacer arms have greater flexibility and reduced steric hindrance.
  • Cleavability: Do you require a crosslinker that is cleavable or reversible? A cleavable crosslinker is recommended if you need to dissociate the linked molecules to recover the individual components? Cleavable crosslinkers are often employed in ‘bait and prey’ analysis.
  • Final analysis method: Are the crosslinked proteins/molecules destined for analysis by gel electrophoresis, western blotting, or mass spectrometry? If eventual analysis will be by mass spectrometry, we offer deuterated analogs of standard ‘light’ crosslinkers for improved identification of components involved in the protein interaction.

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    Ex vivo T lymphocyte expansion for retroviral transduction: influence of serum-free media on variations in cell expansion rates and lymphocyte subset distribution. Citations & References

    • Authors: Carlens S, Gilljam M, Remberger M, Aschan J, Christensson B, Dilber MS,
    • Journal: Exp Hematol
    • PubMed ID: 11027832
    Catalog #

    Anabolic and catabolic responses of human articular chondrocytes to varying oxygen percentages. Citations & References

    • Authors: Strobel S, Loparic M, Wendt D, Schenk AD, Candrian C, Lindberg RL, Moldovan F, Barbero A, Martin I
    • Journal: Arthritis Res Ther
    • PubMed ID: 20193091

    Preferential killing of human lung cancer cell lines with mitochondrial dysfunction by nonthermal dielectric barrier discharge plasma. Citations & References

    • Authors: Panngom K, Baik KY, Nam MK, Han JH, Rhim H, Choi EH
    • Journal: Cell Death Dis
    • PubMed ID: 23703387
    Catalog # M34152

    How do the Total Exosome Isolation reagents work and what makes them different from the other techniques for exosome isolation? Product FAQ

    Answer

    Isolation of exosomes is presently a tedious, non-specific, and difficult process. In the course of development of reagents for isolation of exosomes, we evaluated many different technologies, including “gold standard” ultracentrifugation, ultrafiltration; gel-filtration columns, HPLC, and filters. In addition to these simpler methods, we evaluated more advanced approaches including precipitation using various polymers, and bead and column binding using antibodies and various lectins. We also evaluated commercially available products from System Biosciences and other companies. After evaluation, we selected one of the polymers, based on its superior performance, which became the key component of the Total Exosome Isolation reagents (patent application filed). By tying up water molecules, the reagent forces less-soluble components, such as vesicles, out of solution, which allows them to be collected by a short, low-speed centrifugation. The recovered exosomes are then ready for either biological studies or end-point analysis.

    Answer Id:: E7451

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    Stage-Dependent Effect of TGF-β1 on Chondrogenic Differentiation of Human Embryonic Stem Cells Citations & References

    • Authors: Yang, Z; Sui, L; Toh, WS; Lee, EH; Cao, T
    • Journal: STEM CELLS AND DEVELOPMENT

    How can I amplify my plasmid library in E. coli? Product FAQ

    Answer

    Plasmid libraries may be amplified using several different methods. Growth in liquid culture will frequently result in skewed representation of clones due to differential growth characteristics of individual clones. To minimize these representational biases, we recommend semi-solid amplification. With this method, colonies are grown in 2X LB broth and 0.3% agarose in suspension, facilitating equal growth of all clones and avoiding disproportionate amplification.

    Alternatively, amplification of plasmid libraries may be done on selective agar plates, as described below. Libraries containing expression vector plasmids such as pSPORT or pCMV SPORT should be grown under noninduced conditions (no lactose, no IPTG). The lac repressor gene encoded on the pSPORT plasmid is sufficient to prevent expression of the recombinant genes cloned into the multiple cloning site. Repression of expression is necessary to ensure the amplification of clones whose gene products may be harmful to survival of the E. coli host.

    Recommended protocol for Library Amplification on Plates:
    1. After titering the original library, plate the cells at a density of ~1 x 10E4 CFU per 100 mm plate or ~0.5 x 10E5 CFU per 150 mm plate. Use selective media containing the appropriate antibiotic.
    2. Grow at 37°C until a thick lawn is visible (~6 to 12 h).
    3. Add 5 ml per 100 mm plate or 15 ml per 150 mm plate of S.O.C. medium or LB broth.
    4. Gently coax the bacterial lawn into the broth with a cell scraper (or a glass slide).
    5. Collect the suspension and repeat steps 3 and 4 once more. Pool the broths, then incubate at 37°C for no longer than 1 h with constant swirling.
    6a. If the bacterial suspension is highly dense, add glycerol to a concentration of 15% to 50%; aliquot the cells into cryovials; and immediately store them at -70°C.
    6b. If the bacterial solution is too dilute, centrifuge at 7,000 x g for 10 min and resuspend in a smaller volume of 15% to 50% glycerol/media and freeze.
    7. For use, thaw the cryovials and plate dilutions onto selective media.

    NOTE:
    -The viability of E. coli stored at -70°C will not change substantially for years, unless thawed and refrozen.

    Answer Id:: E4149

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    What factors can contribute to rapid cell death/culture failure? Product FAQ

    Answer

    There are a number of events that can contribute to this:

    1. Incorrect CO2 levels-monitor the level of CO2 manually with a Fyrite kit, available from Bacharach (http://www.bacharach-inc.com/fyrite_analyzers.htm). Check if the manual readings concur with the readings displayed on the incubator. If the incubator has a trace readout, check the printout for fluctuations in CO2 level. Check the settings to insure that CO2 levels are set at appropriate levels for your cell line (usually between 5 and 10%). Check line connections frequently for leaks. Avoid frequent opening and closing of incubator doors.
    2. Temperature fluctuations in the incubator-Monitor the temperature of incubator with a good thermometer inside the incubator.
    3. Fungizone or other preventive antibiotics/antimycotics are present at toxic concentrations-use at recommended levels.
    4. Humidity is incorrect-check the water level in the water pan. Humidity is vital to appropriate gas exchange for many types of cells and media (i.e., appropriate CO2 levels are largely irrelevant for most cultures if the humidity level is not high enough).
    5. Incorrect osmotic pressure in medium-check osmolality of complete medium. Most mammalian cells can tolerate an osmolality of 260 to 350 mOsm/kg. Additions of reagents such as HEPES and drugs may affect osmolality.
    6. Contamination by microorganisms-bacterial and fungal contaminations are usually easily visible; symptoms of mycoplasma contamination are more subtle, and careful monitoring of culture morphology and regular testing are necessary to detect this type of contamination.
    7. Inappropriate medium is being used-double-check that the medium used is appropriate for your cell type and culture application. For example, ensure that the medium being used for serum-free culture is actually designed for serum-free culture; make sure that appropriate selective drugs are used at appropriate levels; check the expiration dates for the reagents being used; and store medium at appropriate temperatures in the dark.

    Answer Id:: E11905

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    What kind of product do you suggest if I would like to use Dynabeads magnetic beads to isolate exosomes? Product FAQ

    Answer

    We have exosome isolation kits for Exosome-Human CD63 (Cat. No. 10606D), Exosome-Human CD9 (Cat. No. 10614D), Exosome-Human CD81 (Cat. No. 10616D), and Exosome-Human EpCAM intended for isolating exosomes with these commonly used exosome surface antigens. If you are interested to isolating exosomes with other specific surface markers using your own antibody, you can use our Dynabeads exosome immunoprecipitation (Protein A, Cat. No. 10610D), Dynabeads exosome immunoprecipitation (Protein G, Cat. No. 10612D), or Exosome-Streptavidin for isolation/detection (Cat. No. 10608D). In addition, anti-mouse IgG Dynabeads magnetic beads (Cat Nos. 11031 or 11033) also can be employed in exosome isolation using mouse monoclonal antibodies against selected surface markers.

    Answer Id:: E12124

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    The differentiation of mesenchymal stem cells by mechanical stress or/and co-culture system. Citations & References

    • Authors: Lee IC, Wang JH, Lee YT, Young TH
    • Journal: Biochem Biophys Res Commun
    • PubMed ID: 17107659

    Leptin treatment of in vitro cultured embryos increases outgrowth rate of inner cell mass during embryonic stem cell derivation. Citations & References

    • Authors: Taskin AC, Kocabay A, Ebrahimi A, Karahuseyinoglu S, Sahin GN, Ozcimen B, Ruacan A, Onder TT
    • Journal: In Vitro Cell Dev Biol Anim
    • PubMed ID: 31214928
    Catalog # H1398

    GTSF-2: a new, versatile cell culture medium for diverse normal and transformed mammalian cells. Citations & References

    • Authors: Lelkes PI, Ramos E, Nikolaychik VV, Wankowski DM, Unsworth BR, Goodwin TJ
    • Journal: In Vitro Cell Dev Biol Anim
    • PubMed ID: 9196892
    Catalog # R12204

    Characterization of free-floating spheres from human trabecular meshwork (HTM) cell culture in vitro. Citations & References

    • Authors: Gonzalez P, Epstein DL, Luna C, Liton PB
    • Journal: Exp Eye Res
    • PubMed ID: 16310191

    T cell differentiation/maturation of CD34+ stem cells from HIV-seropositive hemophiliacs in cultured thymic epithelial fragments. Citations & References

    • Authors: Ruiz M, Roodman ST, Bouhasin JD, Knutsen AP
    • Journal: Stem Cells
    • PubMed ID: 8820959
    Catalog # C2927
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