Anti cd4 percp cy5

The following antibodies were used for flow cytometry: anti-CD4 PerCP/Cy5.5 (Clone GK1.5, BioLegend), anti-CD3 APC-Cy7 (Clone 145-2C11, BD Biosciences), anti-TNF-α PE-Cy7 (Clone MP6-XT22, BD Biosciences), anti-IFN-γ AF700 (Clone XMG1.2, BD Biosciences), anti-IL-2 APC (Clone JES6-5H4, BD Biosciences).

For mouse cell phenotype analysis, anti CD3-percp/cy5.5, anti CD4-FITC, anti CXCR5-allophycocyanin, anti ICOS-PE, and anti PD-1-PE were purchased from BioLegend (San Diego, CA, USA). Anti CD19-FITC, anti CD138-PE, anti IgD-allophycocyanin, anti CD27-percp/cy5.5, and relevant IgG isotypes were purchased from eBioscience (San Diego, CA, USA).
For human cell analysis, anti CD3-FITC, anti CD4-percp/cy5.5, anti CXCR5-allophycocyanin, anti PD-1-PE, and anti CD19-PE were purchased from BioLegend. Anti-IL-21 neutralizing antibody (eBioscience) and anti-CD40 (BioLegend) were used for functional analysis in vitro.

Autologous T cells were co-cultured with matured DCs for 5 days at a 10:1 ratio, after being submitted to a staining protocol with carboxyfluorescein succinimidyl ester (CFSE). All co-cultures were carried out in U-bottomed 96-well plates in a final volume of 200 µL of RPMI medium. At the end of the co-culture period, cells were stained with fluorescence-conjugated antibodies, namely CD4-PerCP/Cy5.5 and CD8-APC antibodies (BioLegend) in order to evaluate the percentage of positive T cell proliferation. Type 1 T cells (Th1), type 2 T cells (Th2), and regulatory T cells (Treg) subsets were also evaluated through flow cytometry after the co-culture period with DCs for 5 days. The autologous T cells were stained using anti-CD4-PerCP/Cy5.5, anti-CD8-APC, anti-CD25-APC, anti-forkhead-box-P3 (FoxP3)-FITC, anti-GATA-binding protein 3 (GATA3)-FITC, and anti-T-box protein expressed in T cells (T-bet)-PE (Biolegend). As some markers are intracellular, the Cyto-Fast™ Fix/Perm Buffer Set (BioLegend), a fixation and cell permeabilization kit, was used for the intracellular staining, according to the manufacturer’s instructions. Data were analysed with GraphPad Prism version 8 (GraphPad Software, San Diego, CA, USA) and the results are presented as a percentage of positive cells (%) after subtraction of isotype control values.

Ko143 and LPS (Eschericia coli strain O111:B4) were obtained from Sigma Chemical Co (St. Louis, MO). Isotype control antibodies (Abs) (IgG1, IgG2a and IgG2b), anti-ABCG2-FITC (IgG2b, 5D3), anti-CD1c-APCcy7 (IgG1, L161), anti-CD4-PerCP/Cy5.5 (IgG2b, OKT4), anti-CD8-Pacific Blue (IgG1, HIT8a), anti-CD11c-APC (IgG1, 3.9), anti-CD123-PEcy7 (IgG1, 6H6), anti-CD83-FITC (IgG1, HB15e), anti-CD25-APC and -PEcy7 (IgG1, M-A251), anti-FOXP3-Pacific Blue and -Alexa Flour-488 (IgG1, 206D), anti-IL-10-PE (IgG2a, JES3-19F1) and anti-IFN-γ-Alexa Flour-488 (IgG1, 4S.B3) Abs were obtained from Biolegend; and neutralizing Ab against human IL-10 (IgG2a, JES3-19F1) was purchased from Biolegend. Abs against phosphorylated form of p38, AKT, ERK and phospho-IKK were obtained from Cell Signaling Technology (Beverly, MA).

For flow cytometry analysis, isolated brain cells were pre-incubated for 15 min with purified anti-mouse CD16/32 (BioLegend, San Diego, CA, USA) to block Fc-mediated non-specific binding of antibodies. Then, cells were stained with the following antibodies: anti-CD45-BV421, anti-CD3ε-PE/Dazzle 594, anti-CD19-PE, anti-CD4-PerCP/Cy5.5, anti-CD8a-PE/Cy7, anti-CD69-APC/Cy7, anti-H-2-FITC, anti-CD317-PE, anti-Ly6C-APC-Cy7, anti-I-A/I-E-BV605 (all from BioLegend), anti-CD11b-AF700 (BD Biosciences, Franklin Lakes, NJ, USA), anti-CD45R/B220-APC (Thermo Fisher Scientific), and anti-CD11c-PE/Cy7 (Tonbo Biosciences, San Diego, CA, USA) for 20 min on ice. After surface staining, dead cells were stained with Zombie Aqua™ Fixable Viability Kit (BioLegend).
Data were acquired on a FACS LSR Fortessa (BD Biosciences), and the percentage of each cell population and mean fluorescence intensity were analyzed using FlowJo software (TreeStar Inc., Ashland, OR, USA). For the analysis of cell numbers of a specific population, total brain cells were visually counted after density gradient centrifugation as described above; then, actual numbers were calculated using the percentages from flow cytometric analysis.