Reagent List
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Important Note
The Human CD3/CD28 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 Human CD3/CD28 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.
The Human CD3/CD28 T Cell Activation Beads are at a concentration of 4E+07 particles/mL
Note: Ensure that all steps before harvesting the cells are done in sterile conditions under a Biosafety Cabinet.
Prepare Cells
- Use Lymphopure (Cat. Nos.# 426201/426202) when isolating PBMCs from anti-coagulated whole blood by density centrifugation. Isolate CD3 T cells by using the MojoSort™ Human CD3 T Cell Isolation Kit (Cat. Nos. #480021/480022/480131).
- Prepare cell culture medium (e.g. RPMI-1640 with 2 mM L-Glutamine, 10% Fetal Bovine Serum (FBS), and 1% Penicillin/streptomycin).
Wash Human CD3/CD28 T Cell Activation Beads
- Thoroughly resuspend the Human CD3/CD28 T Cell Activation Beads by vortexing for 30 sec. Ensure that there is no visible pellet at the bottom of the vial before proceeding.
- Transfer the desired volume of Human CD3/CD28 T Cell Activation Beads to a sterile 12 x 75 mm (5mL) tube.
- Add 1 mL of culture medium and mix thoroughly. Ensure that the entire inside surface of the culture tube is coated in medium.
- Place the tube into a MojoSort™ Magnet. Incubate at RT for 1 min.
- Without removing the tube from the magnet, discard the supernatant by gently pouring from the tube.
- Remove the tube from the magnet and resuspend in culture medium.
Activating T Cells using Human CD3/CD28 T Cell Activation Beads
- Start with 8 x 104 cells in 100-200 µL of cell culture medium in a 96-well tissue culture plate.
- Add pre-washed Human CD3/CD28 T Cell Activation Beads to the cells at a 1:1 bead-to-cell ratio.
- Incubate in a humidified CO2 incubator at 37oC until the activated T cells are ready for harvest.
- For flow cytometry applications, remove the beads from the activated T cells by transferring the cells to a sterile 12 x 75 mm (5 mL) 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.
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