Human CD3/CD28/CD137 T Cell Activation Beads Protocol

Reagent List

Human CD3/CD28/CD137 T Cell Activation Beads MojoSort™ Buffer (5X) (Cat. No. 480017), sterile filtered upon dilution to 1X MojoSort™ Magnet (Cat. No. 480019/480020/480173) or compatible magnetic separation system Human T Cell Isolation Kits such as: MojoSort™ Human CD3 T Cell Isolation Kit (Cat. No. 480021/480022/480131) MojoSort on Columns™ Human CD3 T Cell Isolation Kit (Cat. No. 480194) Adjustable pipettes Serological pipet

Sterile 5 mL (12 x 75 mm), 14 mL (17 x 100 mm), or 50 mL (30 x 115 mm) polypropylene tubes Reagents for cell preparation and culturing (medium, plates, flasks, incubator, etc.)

Important Note

• Activation Beads are at a concentration of 4E+07 particles/mL.
• The Human CD3/CD28/CD137 T Cell Activation Beads can be used with other commercially available MojoSort™ magnetic separators. Please contact BioLegend Technical Service for more information and guidance.

 

Protocol

Product description and procedure summary:

This protocol provides instructions for activating and expanding human T cells using our Human CD3/CD28/CD137 T Cell Activation Beads. After washing the beads, simply mix them together with your T cells at a 1:1 particle-to-cell ratio and incubate them in a humidified CO₂ incubator for robust stimulation and expansion. Downstream applications include functional assays, gene expression analysis, and phenotypic characterization.

Note: Ensure that all steps before harvesting the cells are done in sterile conditions under a Biosafety Cabinet.

 

Prepare Cells

 

1. Use Lymphopure™ (Cat. Nos. 426201/426202) when isolating PBMCs from anti-coagulated whole blood by density centrifugation. Isolate CD3 T cells by using a kit such as the MojoSort™ Human CD3 T Cell Isolation Kit (Cat. Nos. 480021/480022/480131) or MojoSort on Columns™ Human CD3 T Cell Isolation Kit (Cat. No. 480194).
2. Prepare cell culture medium (e.g. RPMI-1640 with 2 mM L-Glutamine, 10% Fetal Bovine Serum (FBS), and 1% Penicillin/Streptomycin).

 

Wash Activation Beads

 

1. Thoroughly resuspend the activation beads by vortexing for 30 sec. Ensure that there is no visible pellet at the bottom of the vial before proceeding.
2. Transfer the desired volume of Human CD3/CD28/CD137 T Cell Activation Beads to a sterile tube.
3. Add 1 mL of culture medium and mix thoroughly. Ensure that the entire inside surface of the culture tube is coated in medium.
4. Place the tube into a MojoSort™ Magnet. Incubate at RT for 1 min.
5. Without removing the tube from the magnet, discard the supernatant by gently decanting from the tube.
6. Remove the tube from the magnet and resuspend beads in culture medium.

 

Activating T Cells using Human CD3/CD28/CD137 T Cell Activation Beads

 

1. Start with 8 x 10⁴ T cells in 100-200 µL of cell culture medium in a 96-well tissue culture plate.
2. Add pre-washed activation beads to the cells at a 1:1 bead-to-cell ratio.
   Note: Activation Beads are at a concentration of 4E+07 particles/mL.
3. Incubate in a humidified CO₂ incubator at 37°C until the activated T cells are ready for harvest.
4. For flow cytometry applications, you may remove the beads from the activated T cells by transferring the cells to a sterile tube and placing the tube in a MojoSort™ Magnet for 1 minute at RT. Pour out the supernatant with the activated T cells into a new tube; the initial tube containing the beads can be discarded.
   Optional: User may perform this step with cells in pre-warmed culture media (37oC) or desired buffer.