AcnoviaBeads Human T-Activator CD3/CD28 (Cat. No. AC68951) are intended for human T cells separation and in vitro expansion.  This can been applied to CAR-T and other T cell culture technologies application. AcnoviaBeads Human T-Activator CD3/CD28 is composed of 4.5 μm magnetic beads conjugated with anti-human CD3 and anti-human  CD28 antibodies，and offers a simple method for isolation and expansion of human T cells. First，AcnoviaBeads Human T-Activator  CD3/CD28 enables easy separation and concentration of CD3⁺ T cells from PBMCs. After isolation, covalent binding of anti-CD3 and antiCD28 antibodies on magnetic beads provide both the primary and co-stimulatory signals required to regulate T cell activation and  expansion. During cell expansion, the cell culture requires additional addition of other cytokines, such as human IL-2, IL-7, and IL-15 for  more efficient activation. The activated cell can be expanded 1000-fold over a 10-13 day culture period.

Catalog:AC68951

Reactivity:Human

Concentration:2×10⁸ beads/mL

Particle size:4.5 μm

Endotoxin:<1 EU/mL

Usage:in vitro T cell activation and expansion in enriched T cell or PBMCs 

Formulation:phosphate buffered saline (PBS), containing Human Serum Albumin (HSA), pH 7.4

Stability:24 months 

Storage:2 ℃ to 8 ℃, Do not freeze

Cat. No.AC68951-1

Name :AcnoviaBeads Human T-Activator CD3CD28

Size:1 mL

Capacity:For isolation: 6.6×10⁷ CD3⁺ T cells;For activation: 2×10⁸ enriched CD3⁺ T cells ;

Cat. No.AC68951-1

Name :AcnoviaBeads Human T-Activator CD3CD28

Size:10ml

Capacity:For isolation: 6.6×10⁸ CD3⁺ T cells;For activation: 2×10⁹ enriched CD3⁺ T cells ;

Materials Required
• Human T cell culture media.  
• Human cytokines for optimal expansion, such as, IL-2, IL-7, IL-15.

Wash AcnoviaBeads Human T-Activator CD3/CD28  

1. Resuspend the AcnoviaBeads Human T-Activator CD3/CD28 (beads) in the tube (vortex for >30 sec, or tilt and rotate for 5min).

2. Transfer the beads with a desired volume into a tub.

3. Add equal volume of PBS (containing 1% HSA). If the beads volume is less than 1mL, add 1mL PBS (containing 1% HSA) for resuspension.

4. Place the tube on the magnet for 1min, and then discard the supernatant.

5.Remove the tube from the magnet, then use the same volume of PBS (containing 1% HSA) to resuspend the beads.

Separate CD3⁺ T cells

1. For Ficoll isolated PBMCs, gently resuspend the cells in PBS (containing 1% HSA), and adjust the cell density to 2-5×10⁷ cells/mL. Note that the total number of cells should not exceed 2×10⁸ cells/mL.

Note: Before you begin, determine the percentage of CD3⁺ T cells in the sample by flow cytometry.

2. Add washed beads into the PBMCs in a ratio of 3 (beads):1  (CD3⁺ T cell),  if the cells are pure isolated T cells, the ratio of  beads to cells is adjusted to 1:1.

3. Rotate the samples with a speed of 50~120 rpm/min, and incubate them at room temperature for 30 min.

4. Dilute the mixture of beads and cells with T cell culture media or PBS  (containing 1% HSA) to ensure the separation volume for magnetic selection.Following this, place the tube on the magnet for 1~2min.

5. Remove the supernatant, then resuspend the mixture of beads and cells with T cell culture media containing 300 IU/mL IL-2, and adjust the cell density to 0.5×10⁶ cells/mL~1×10⁶ cells/mL.

6.Put the cell suspension in the incubator with 37 ℃, 5% CO₂.

T cell activation and expansion

1.  Count the number of T cells daily, beginning on day 3 of culture.

2. At the later stage of cell expansion, gently blow the cell suspension in the culture regularly to dissociate the beads and cells. 

3. When CD3⁺ T cells >1×106 cells/mL, add  T cell culture media containing IL-2 to dilute cells, and adjust the cell density to about 0.5×105 cells/mL.

4.At the end of culture (day 9-14), count the cells and remove the beads with magnets.