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Cell-Vive™ T-NK Xeno-Free Serum Substitute, GMP

420502

50 mL

206 USD

420502

Cell-Vive™ T-NK Xeno-Free Serum Substitute, GMP

Xeno-free, Serum-free Human Serum Replacement

50 mL

206 USD

CV

Note: This product contains human-derived components.Complete cell Culture Media Preparation:Thaw Cell-Vive™ T-NK Xeno-Free Serum Substitute, GMP overnight at 4°C. The product can be stored at 4°C in the dark up to 2 weeks post-thaw. Do not refreeze after initial thaw. 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-NK Xeno-Free 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-NK Xeno-Free Serum Substitute, GMP can be used with tissue culture plates, flasks and the G-REX® system. If using a bag format, testing is recommended with the specific bag being used.Cell Activation methods:Cell-Vive™ T-NK Xeno-Free Serum Substitute, GMP can be used in combination with anti-human CD3 and CD28 antibodies in solution, plate-coated or conjugated to magnetic beads. 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-NK Xeno-Free Serum Substitute, GMP and 200 IU/mL recombinant human IL-2 (BioLegend, Cat. No. 791906 or equivalent) for T cell culture, and 1000 IU/mL for NK 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.;PBMC-Derived NK cell culture protocol using K562 feeder cells:;Start by inactivating K562 cells with mitomycin-C (20 µg/mL in K562 growth media) for 45 min at 37°C and 5% CO2.;Spin down inactivated K562 cells at 300xg for 5 min. Wash cell pellet twice with fresh media, to remove any leftover mitomycin-C.;Equilibrate appropriate amount of complete media at 37°C for 10-15 min.;Plate PBMC (fresh or frozen) at 0.5 x 10^6 cells/mL and incubate at 37°C and 5% CO2.;Add feeder cells (inactivated K562 cells (recommended value of 1 K562 cell to 2 PBMC cells; optimal value can be determined for desired culture setup).;Allow NK cells to get activated for 2-3 days.;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. Re-feed NK cell culture with freshly inactivated K562 cells at day 7.;Repeat steps 4-6 every 2 days up to day 14 (or appropriate culture end).;Follow with further processing and/or cell analysis.;G-REX® is a registered trademark of Wilson Wolf Corporation.Parafilm is a registered trademark of Bemis Company, Inc.;