Babco, Signet, Sternberger Monoclonals, Senetek, Covance

other

Cell-Vive™ T cell CD Serum Substitute, GMP

420506

50 mL

347 USD

420506

Cell-Vive™ T cell CD Serum Substitute, GMP

Chemically-Defined (CD), Serum-free Human Serum Replacement

50 mL

347 USD

CV

Complete cell Culture Media Preparation:Thaw Cell-Vive™ T cell CD Serum Substitute, GMP overnight at 4°C. The product can be stored at 4°C in the dark for up to 2 weeks post-thaw. Do not refreeze after initial thaw. The presence of cloudiness in the product after thaw is normal and relates to the high enrichment of the product. Make sure to mix product prior to use. Once the product is diluted into basal media, no cloudiness should be observed. Dilute Cell-Vive™ T cell CD Serum Substitute, GMP to 5% v/v in IMDM and 200 IU/mL of recombinant human IL-2 (BioLegend, Cat. No. 791906 or equivalent). Alternatively, the product can be used in combination with your basal media of choice and cytokines of interest. To determine optimal performance, a titration of the product under your desired cell culture setup may be required.Cell Culture Platform:Cell-Vive™ T cell CD Serum Substitute, GMP can be used with tissue culture plates, and flasks. If using a bag format, testing is recommended with the specific bag being used.Cell Activation methods:Cell-Vive™ T cell CD Serum Substitute, GMP can be used in combination with anti-human CD3 and CD28 antibodies in solution, plate-coated. Optimal values should be determined if using a different culture setup.For the protocols below, complete media refers to IMDM with 5% v/v Cell-Vive™ T cell CD Serum Substitute, GMP, and 200 IU/mL recombinant human IL-2 (BioLegend, Cat. No. 791906 or equivalent) for T cell culture.;1. Coating plates with anti-human CD3 and CD28 antibodiesAdd 1 µg/mL of anti-human CD3 (OKT3 clone, BioLegend, Cat. No. 317326 or equivalent) and 1 µg/mL of anti-CD28 antibodies (CD28.2 clone, BioLegend, Cat. No. 302934 or equivalent) to PBS. Add solution to the vessel, at recommended volume (table below). Incubate for 2 hrs at 37°C in a tissue culture incubator (or overnight at 4°C, preventing evaporation with Parafilm®). Aspirate solution and wash twice with 1 volume of PBS. Wells are ready to be used. Do not let wells dry.TABLE 1: Recommended volumes of coating solution composed of anti-human CD3 and CD28 antibodies.;Vessel;Recommended volume;;12- well plate;0.75 mL;;6- well plate;2 mL;;T25 flask;5 mL;;T75 flask;15 mL;;2. Soluble antibodiesAdd 10 µg/mL of anti-human CD3 (OKT3 clone, BioLegend, Cat. No. 317326 or equivalent) and 5 µg/mL of anti-CD28 antibodies (CD28.2 clone, BioLegend, Cat. No. 302934 or equivalent) to complete media.PBMC-Derived T cell culture protocol:;Equilibrate appropriate amount of complete media at 37°C for 10-15 min.;Plate PBMC (fresh or frozen) at 1 x 10^6 cells/mL and incubate at 37°C and 5% CO2.;Allow cells to get activated for 3-4 days.;At day 3-4, dissociate cell clumps by pipetting until a single cell solution is observed.;Determine cell density.;Dilute cell culture to 0.25 - 0.5 x 10^6 cells/mL and incubate at 37°C and 5% CO2.;Repeat steps 4-6 every 2 days. Monitor cell culture to make sure confluency is not reached. Pending donor-to-donor variability exponential expansion may require more frequent feeding. Complete media has been shown to support up to 4 x 10^6 cells/mL without impact on cell viability and quality.;Follow with further processing and/or cell analysis.;Parafilm is a registered trademark of Bemis Company, Inc.;