Dear Penny!

You look at this articles:

Article Comparison of anti-CD3 and anti-CD28-coated beads with solub...

Preparation of anti-CD3/CD28 beads

To prepare antibody-coated beads of varying composition, streptavidin-labeled beads were coated with varying mixtures of biotinylated anti-CD3 and anti-CD28 antibodies. To this end, streptavidin-M280 beads were washed once with sterile PBS/BSA and resuspended at 10-50 millions beads/ml. Preliminary dose response studies, using FITC-labeled anti-mouse IgG and flow cytometry to monitor biotinylated antibody binding to beads, established that beads were saturated by 100 ng of biotinylated antibody/million beads. Consequently this total immunoglobulin/bead ratio was routinely used for bead coating. To vary the ratio of antibody coating on beads equimolar solutions of anti-CD3 and anti-CD28 were mixed at 1:0, 1:5, 1:10, 1:20, 1:40, 1:80, 2 1:160, and 0:1 ratios. Control beads were coated with biotinylated IgG1 isotype. Coating was performed on a rotator stand at room temperature for 2-3 hours. Beads were then washed two times with filtered PBS/BSA, once with complete medium, and then resuspended in RPMI 1640 complete medium. Antibody coating was performed as needed, but preliminary studies established that beads could be stored 4°C for at least one week without any change in potency. In selected studies, T cells were also stimulated using commercially prepared anti-CD3 and anti-CD28 coated beads (CD3/CD28 T cell Expander, Invitrogen).

Flow cytometry

Flow cytometry was performed using a 4-color Facscalibur (BD biosciences). The standard phenotypic analysis was performed at different time point using antibody panels as described in the results. The flow data was analyzed using Flow-Jo software.

Article T cell stimulation and expansion using anti-CD3/CD28 beads

Thanks Alexandr,

The article focuses more on T cell activation. I would like to activate T cells but then look at T-cell dependent B cell responses.

Penny