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MB Sample ID: SA343034
| Local Sample ID: | WT CD19-BBz CART_1 |
| Subject ID: | SU003285 |
| Subject Type: | Cultured cells |
| Subject Species: | Mus musculus |
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Collection:
| Collection ID: | CO003278 |
| Collection Summary: | Mouse CD8 cells were isolated with negative selection as described above, activated with anti-CD3/anti-CD28 beads (Dynabeads™ Mouse T-Activator CD3/CD28 for T-Cell Expansion and Activation, Gibco, Cat#11453D) with addition of recombinant human IL-2 (rhIL-2) (60 IU/ml) and human recombinant IL-7 (10 ng/ml). On the second and third day of activation, the supernatant containing the CD19-BBz CAR/hEGFR retrovirus was spun on a plate coated with retronectin (Takara Bio Inc.) (2000xg for 2.5h at 32C). The activated cells were added to the viral-coated plate for transduction. The transduced CD8 CAR-T cells were removed from the beads on the fourth day and expanded with rhIL-2 (60 IU/ml) or rhIL-7 (10 ng/ml) and rhL-15 (100 ng/ml). After 2 days (1st expansion) cells were split 1/3 with fresh cytokine-containing medium. After 2 more days (2nd expansion), cells were split again with fresh cytokine-containing medium. Two days later (3rd expansion) cells were used for experiment or incubated in cytokine free-medium. For CAR+ cells enrichment, the CAR-T cells expanded with IL-2 for 3 expansions were incubated with an anti-hEGFR-PE (BioLegend, Cat#352904, RRID: AB_10896794) antibody, followed by incubation with anti-PE microbeads (Miltenyi, order#130-048-801), and purified using the Miltenyi LS columns as recommended by the manufacturer (Miltenyi). The enrichment resulted in a 98-100% CAR+ population, as verified by flow cytometry. |
| Sample Type: | T-cells |
