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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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FreeStyle F17 Expression Medium was designed from a different base formulation than FreeStyle 293 Expression Medium. FreeStyle F17 medium shows equal or better performance than the FreeStye 293 medium for certain cell lines and proteins. It is capable of supporting the growth of 293 and CHO cells and offers a wider variety and selection of transient cell culture media options.

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There are several publications using HPLM. A list of select publications using HPLM can be found on the HPLM web page: https://www.thermofisher.com/us/en/home/life-science/cell-culture/mammalian-cell-culture/classical-media/human-plasma-like-medium.html

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Both VP-SFM (Cat. No. 11681020) and OptiPro SFM (Cat. No. 12309019) can be used to culture Vero cells and will provide equivalent cell growth. VP-SFM is serum-free but not protein-free. It contains human recombinant insulin in its formulation. The human recombinant insulin uses animal origin component in its manufacturing process. OptiPRO SFM is serum-free, protein-free, and animal origin free. Both products have a drug master file associated with them.

If you will be choosing either of these formulations, we would also recommend using TrypLE Express (Cat. No. 12605010 or 12604013) or TrypLE Select (Cat. No. 12563011) reagent for passaging the cells. TrypLE Express and Select reagents contain the same enzyme. The main difference is in the manufacturing process. TrypLE Select reagent is made in disposable or dedicated equipment that does not come in contact with animal origin components. TrypLE Express reagent can be manufactured in vessels that may have been used in the manufacture of cell culture media or reagents that contain animal-origin components.

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We recommend that you discard the culture and begin the process again. You will want to select and maintain a high quality of PSCs before inducing neural differentiation.

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Unlike other methods that require multiple components and take five or more days, the PSC Definitive Endoderm Induction Kit can generate ?90% CXCR4+/PDGFRalpha- definitive endoderm cells with only two ready-to-use media in just two days. Each medium is supplied as a 1X complete medium, requiring no mixing of additional components, and resultant definitive endoderm is capable of differentiating to a wide range of definitive endoderm derived cells including mid/hindgut, liver, and pancreas.

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

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FreeStyle 293-F cells are derived from HEK293 cells; they were just cloned extensively for growth, viability, and protein production in the new serum-free FreeStyle 293 medium. Thermo Fisher Scientific coined the name “FreeStyle” to signifiy the freedom of switching the cells to the defined serum-free FreeStyle media system.

The parental HEK293 cells, if adapted to the same media as FreeStyle 293F cells, should have the same transfection efficiency. However the final protein production levels may be very different as the FreeStyle 293F cells have been cloned and selected for maximal protein production.

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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.

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We do not recommend freezing the media.

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When cells meant to be grown in suspension are grown in static culture, they may form clumps. These clumps will severely limit transfection efficiency and protein expression. It is suggested that FreeStyle 293 cells in FreeStyle media and CHO-S cells in CD-CHO or CHO-SFM are grown in agitated suspension to reduce the appearance of clumps. However, if clumps do form, you can try the following protocol to select for cells that don't form clumps:

- Transfer cells into an appropriate size centrifuge tube that will hold the entire cell suspension.
- Allow cells to sit undisturbed for about 5 minutes. The time can vary depending on the specific cell line. Larger cell clumps will settle to the bottom of the tube.
- Collect cells from the upper portion of the tube to passage into a new flask.

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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.

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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.

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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.

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Thermo Fisher Scientific offers multiple serum free media (SFM) and chemically defined (CD) media options for CHO cultures. For adherent cultures, CD CHO (10743-011) and CD OptiCHO medium (12681-011) are robust media; both are protein free and animal origin free (AOF). For adherent cultures, CD CHO-A (097-0182DJ) is a widely used protein free and animal origin free medium. There are other formulations available to suit other research needs; contact Technical Support for further information.

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