Pe anti human cd28

From peripheral blood samples collected of RA patients and CS, peripheral blood mononuclear cells (PBMC) were obtained by Lymphoprep density‐gradient centrifugation. PBMC were washed with phosphate‐buffered saline supplemented with 0.5% bovine serum albumin. Their viability was evaluated using the trypan blue exclusion method, and only those samples with more than 90% of viability were considered.
Multicolor flow cytometry was used to analyze from PBMC the expression of CD28, CTLA‐4 on gated CD4+, and CD8 + T cells. Cell surface staining was performed with fluorochrome‐labeled monoclonal primary antibodies purchased from eBioscience: FITC anti‐human CD3, PE/Cy5 anti‐human CD8, PE anti‐human CD28, and PE anti‐human CD152. Corresponding isotype control antibodies were also included from eBioscience.
Assay tubes were stained with a mixture of corresponding antibodies at the recommended dilution and incubated for 20 minutes at room temperature in the dark. After incubation, the cells were washed and fixed in PBS containing 1% paraformaldehyde. Finally, data were acquired using EPICS XL‐MCL flow cytometer (Beckman‐Coulter) and cell surface expression of CTLA‐4 and CD28 on CD3CD4 + and CD3CD8 + was analyzed with WinMDI 2.8 software (Scripps Institute, La Jolla, CA).

Anti-mouse CD3 (clone 145-2C11) and anti-CD28 (clone PV-1) antibodies were prepared in our laboratory. DMEM, penicillin and streptomycin from GIBCO Inc. (Grand Island, NY, USA); fetal bovine serum (FBS) from HyClone Inc. (Logan, UT, USA); anti-mouse antibodies including Alexa 488 anti-Foxp3, -IFNγ and -perforin, PE anti-CD25, -CD39, -CD101, -CTLA-4, -granzyme B, ICOS and -IL-2, PerCP-Cy5.5 anti-CD73 and APC anti-CD45.2, FITC anti-human CD8, PE anti-human CD28, APC anti-human CD39 and PE-Cy7 anti-human CD73 from eBioscience (San Diego, CA, USA); PE anti-TNFα, -CCR6, -CD103, -Galectin-9 and -Helios antibodies from BioLegend (San Diego, CA, USA); PE anti-CD122, -CTLA-4, -LAG3 and -PD-1, APC anti-FR4, and PE-Cy7 anti-GITR antibodies from BD Bioscience (San Jose, CA, USA); Anti-CD73 antibody from Abcam (Cambridge, MA, USA); Mitomycin C, LPS, MTT, α, β-methylene adenosine 5’-diphosphate (AMPCP) from Sigma (St. Louis, MO, USA); KC7F2 from Calbiochem (Burlington, MA, USA); ZM241385 and MRS1754 from Tocris (Minneapolis, MN, USA); CFSE from Molecular Probes (Eugene, OR, USA); M2 peptides (LCMV gp33-41, KAVYNFATM) from AnaSpec, Inc. (San Jose, CA, USA); LY294002 from Cell signaling Technology (Danvers, MA, USA) were purchased.