Anti cd28 ab

CD4⁺CD25⁺ T_reg cells were purified from spleen with Miltenyi beads, and cultured in RPMI-1640 (Gibco, Grand Island, NY) supplemented with 10% FBS (Hyclone, Logan, UT), 10 mM HEPES (Sigma, St. Louis, MO), 1 mM sodium pyruvate (Sigma), 50 μM 2-ME (Gibco), 100 U/ml penicillin (Sigma) and 100 μg/ml streptomycin (Sigma). Cells were stimulated with 1ug/ml anti-CD3 Ab and 2ug/ml anti-CD28 Ab (eBioscience) and/or 50U rmIL-2 (Peprotech) for 72 hrs. Frequency and expression level of Foxp3 were measured by flow cytometry.

Sub-populations of T cells (CD28⁺CD95⁺ central memory, CD28⁻CD95⁺effector memory, and CD4⁺CD25⁺Foxp3⁺ regulatory cells) and activated B cells
(CD3⁻CD20⁺CD28⁺) in blood were evaluated.^{1 ,2 (link)} For extracellular surface staining, cell suspensions were
incubated for 30 minutes at 4°C with fluorescein-conjugated mouse anti-human Abs as follows: CD3-FITC (1:40), CD4-FITC
(1:200), CD8-PerCp-Cy5.5 (1:200), CD25-APC, CD28-APC (1:200), CD95-PE (1:200), and CD20-PE (1:200). For intracellular Ab staining,
cell suspensions were incubated at 4°C with fluorescein-conjugated mouse anti-human Abs as follows: IFN-γ-PE (1:200,
30 minutes) and Foxp3-PE (1:200, 1 hour). Intracellular IFN-γ staining was performed after stimulation overnight with
anti-CD3 Ab (2.5 μg/mL) and anti-CD28 Ab (0.25 μg/mL) in the presence of GolgiPlug (brefeldin A; 1 μL/1 mL).
All Abs were purchased from eBioscience (San Diego, CA, USA), except CD3-FITC (from BD PharMingen, San Diego, CA, USA) and
anti-CD3 Ab (U-CyTech, Utrecht, The Netherlands). Data were acquired using a FACSCanto flow cytometer (Becton-Dickinson, Mountain
View, CA, USA) and analyzed using FlowJo software (Tree Star, Ashland, OR, USA) (Supplementary Figure S1). Data were presented as the absolute number of cells per unit volume or percentage of
peripheral blood mononuclear cells.