T Cell Activation

Cell-Vive GMP CD3/CD28 Human T Cell Activation Beads are suitable for in vitro or ex vivo activation and expansion of human T cells for ex vivo cell processing. Superparamagnetic microbeads are coated with anti-CD3 (clone OKT3) and anti-CD28 (clone S20013F) antibodies, providing both the primary and co-stimulatory signals required for the activation and expansion of human T cells without the need for antigen-presenting or feeder cells.

Downstream applications include use in ex vivo cell manufacturing workflows.

Here is an overview of the protocol. After washing beads, simply add the beads directly to isolated T cells or to PBMCs at a 1:1 bead-to-cell ratio for robust T cell activation. This is followed by incubation of cells in a humidified CO₂ incubator for precise stimulation and expansion of T cells. The antibody-coated beads can be removed from the cells with a magnetic separator.

*This product was optimized on isolated CD3+ T cells and PBMCs. Alternate sources of T cells may require protocol optimization.

Human peripheral blood mononuclear cells were isolated from whole blood and plated at 5x10⁵ cells/well in complete IMDM culture medium supplemented with 5% human AB serum and 30 IU/mL recombinant human IL-2 (Cat. No. 791908). On Day 0, cells were unstimulated or incubated with a 1:1 ratio of Cell-Vive™ GMP CD3/CD28 Human T Cell Activation Beads (Cat. No. 422606), RUO Human CD3/CD28 T Cell Activation Beads (Cat. No. 422604), or competitor CD3/CD28 Activation Beads. Cells were cultured at 37°C, 5% CO₂ for 11 days and expanded with fresh media every 2-4 days. Total cell count and fold expansion were measured at indicated timepoints.

Human peripheral blood mononuclear cells were isolated from whole blood and plated at 5x10⁵cells/well in complete IMDM culture medium supplemented with 5% human AB serum and 30 IU/mL recombinant human IL-2 (Cat. No. 791908). On Day 0, cells were unstimulated or incubated with a 1:1 ratio of Cell-Vive™ GMP CD3/CD28 Human T Cell Activation Beads (Cat. No. 422606), RUO Human CD3/CD28 T Cell Activation Beads (Cat. No. 422604), or competitor CD3/CD28 Activation Beads. Cells were cultured at 37°C, 5% CO₂ for 11 days and expanded with fresh media every 2-4 days. On indicated timepoints, cells were collected and surface stained with APC anti-human CD3 clone UCHT1 (Cat. No. 300439), PE anti-human CD4 clone RPA-T4 (Cat. No. 300539), FITC anti-human CD8 clone SK1 (Cat. No. 344704), PE/Cyanine7 anti-human PD1 clone A17188B (Cat. No. 621616), APC/Fire™ 750 anti-human CD69 clone FN50 (Cat. No. 310946), and 7-AAD viability dye (Cat. No. 420404) and analyzed by flow cytometry. The frequency of CD69+ or PD1+ cells is shown gated on viable CD3+CD4+ or CD3+CD8+ T cells.

Natural Killer Cell Activation

Cell-Vive™ GMP NKp46/CD2 Human NK Cell Activation Beads allow for convenient in vitro or ex vivo activation of human NK cells using NKp46/CD2 coated superparamagnetic microbeads. No need for antigen-presenting cells or feeder cells.

Downstream applications include functional assays, gene expression analysis, phenotypic characterization, as well as ex vivo cell manufacturing workflows.

Human peripheral blood mononuclear cells were stimulated with Cell-Vive™ GMP NKp46/CD2 Human NK Cell Activation Beads or a competitor’s equivalent product at a bead-to-cell ratio of 1:2 for 14 days. Cells were cultured in IMDM containing 5% human AB serum, 100 IU/mL of recombinant IL-2 (Cat. No. 791902) and 10 ng/mL of recombinant IL-15 (Cat. No. 570314). Cells were split on day 6 and day 9 of culture and re-seeded at a density of 1.0 x 10⁶ cells per mL. Cells were harvested at day 14 for total cell expansion and phenotype analysis. (Left) Fold expansion of CD56+ cells was calculated by comparing the final percentage (determined by flow cytometry) of CD56+ cells and total cell counts on day 14 with the starting percentage of CD56+ cells in day 0 PBMCs. (Right) Total cell viability at day 14.

Further phenotypic analysis was performed on the CD56+ cells at day 14 showing cells generated using Cell-Vive™ GMP NKp46/CD2 Human NK Cell Beads (magenta) or a competitor’s equivalent product (teal).