Alexa fluor 700 conjugated anti cd3

UUO (n = 6 mice) and sham (n = 6 mice) surgeries were performed, and renal single cells were isolated and analyzed by flow cytometry. Briefly, renal tissue was minced and placed into a cocktail of 0.25 mg/mL Liberase Blendzyme 3 (Roche Applied Science), 20 U/mL DNase I (Sigma-Aldrich) and shaken at 37 °C for 20 min. Subsequently, cells were passed through 40 μm nylon mesh and centrifuged (10 min, 200 g, 4 °C). Cells were stained and analyzed using flow cytometry. The following dyes and antibodies were used: APC-Cy7-conjugated anti-CD45, Alexa Fluor 700-conjugated anti-CD3 (both from BD Pharmingen), PerCP/Cy5.5-labeled anti-CD31, PE/Cy7-labeled anti-F4/80 (both from Biolegend), and Cy3-conjugated anti-α-SMA (Sigma). Stained single cells were resuspended in a staining buffer and immediately analyzed with a Becton Dickinson LSRII flow cytometer (BD Biosciences).

The V450-conjugated anti-CD3, Alexa Fluor®700-conjugated anti-CD3, BV650-conjugated anti-CD4, FITC-conjugated anti-CD4, BV605-conjugated anti-CD11b, BV421-conjugated anti-CD11c, BV650-conjugated anti-CD14, PE-Cy^TM7-conjugated anti-CD14, Alexa Fluor®700-conjugated anti-CD16, BV421-conjugated anti-CD16, PE-CF594-conjugated anti-CD16, phycoerythrin (PE)-conjugated anti-CD19, Alexa Fluor®700-conjugated anti-CD19, Alexa Fluor®700-conjugated anti-CD20, V500-conjugated anti-CD45, BV786-conjugated anti-NKp46, Alexa Fluor®700-conjugated anti-CD56, PE-conjugated anti-CD64, PE-Cy^TM7-conjugated anti-CD86, PerCP-Cy5.5-conjugated anti-CD123, BV711-conjugated anti-CD141, BV786-conjugated anti-PDL2, and PE-CF594-conjugated anti-CD163 antibodies were from BD Biosciences. The APC-Alexa Fluor®750-conjugated anti-HLA-DR antibody was purchased from Beckman Coulter. The PE-conjugated anti-CD1c, APC-conjugated anti-CD303, and APC-conjugated anti-SLAN antibodies were from Miltenyi Biotech. The FITC-conjugated anti-CD192 antibody was purchased from R&D Systems. All antibodies were used according to their manufacturer's recommendations. Antibodies combination for monocytes: CD3, CD11b, CD14, CD16, CD19, CD45, CD56, CD64, CD163, CD192, NKp46, HLA-DR, SLAN, and DC: CD1c, CD3, CD4, CD11b, CD11c, CD14, CD16, CD19, CD20, CD45, CD56, CD86, CD123, CD141, CD303, PDL2, HLA-DR.

PBMCs were suspended in RPMI media (Invitrogen) and rested for 2 hours. One million cells were stimulated in vitro with phorbol 12-myristate 13-acetate and ionomycin or with overlapping peptides from SIV p27 protein for 9 hours at 37°C as previously described (58 (link)). After incubation, cells were washed and immunostained with Alexa Fluor 700–conjugated anti-CD3 (BD Biosciences, SP-234), BV650-conjugated anti-CD4 (BD Biosciences, L200), and PerCyP-Cy5.5–conjugated anti-CD8 (Biolegend, OKT4). Cells were washed, permeabilized using a Cytofix/Cytoperm Kit (BD Biosciences), and intracellularly immunostained with PE-Cy7–conjugated anti–IFN-γ (BD Biosciences, B27) and Alexa Fluor 488–conjugated anti–TNF-α (Invitrogen, Mab11).