2), purchased from BD Biosciences (Stockholm, Sweden), APC-Alexa

2), purchased from BD Biosciences (Stockholm, Sweden), APC-Alexa Fluor 700-conjugated anti-CD107a (H4A3), APC-conjugated anti-IL-7 receptor α-chain (IL-7Rα; R34.34), PE-Texas Red-conjugated anti-CD45RA (2H4), fluorescein isothiocyanate (FITC) -conjugated anti-CD8β chain (2ST8.5H7), purchased from Beckman Coulter (Marseille, France), and Pacific Blue-conjugated anti-CD4 (S3.5) purchased from Caltag Laboratories (Burlingame, CA). The lymphocytes were then washed in phosphate-buffered saline (PBS)

with 0·1% fetal bovine serum, and incubated at 4° for 15 min with the anti-CD27 (1A4CD27) antibody (Beckman Coulter) labelled with Pacific Orange using the Zenon Opaganib molecular weight Pacific Orange Mouse IgG1 Labeling Kit obtained from

Invitrogen (Stockholm, Sweden). Human samples were processed the same day, and NHP samples were processed on a different occasion, but also the same day. The median fluorescence intensity (MFI) of IL-7Rα expression therefore allows a comparison of the intensity of IL-7Rα expression on T cells within each species but not between humans and NHPs. Data acquisition was performed using a FACSAria Flow cytometer (BD Biosciences) and results were analysed with FlowJo software (Tree Star Inc., Ashland, OR). Cytokine production was analysed in frozen PBMCs, which were thawed, rested overnight and stimulated for 6 hr in the presence of brefeldin A (10 mg/ml) purchased from Sigma-Aldrich (Sweden AB, Stockholm, Sweden) either Dichloromethane dehalogenase with medium: RPMI-1640 containing l-glutamine

(2 mm), penicillin (100 IU/ml) and streptomycin selleck (10 mg/ml), 10% heat-inactivated fetal bovine serum (Gibco, Invitrogen), or medium and phorbol 12-myristate 13-acetate (PMA)/ionomycin (25 ng/ml and 1 mg/ml, respectively; Sigma-Aldrich). Cells were then washed in PBS, and stained with cell surface marker antibodies: Pacific Blue-conjugated anti-CD3 (SP34-2), PerCP-Cy5.5 conjugated anti-CD4 (L200; BD Biosciences), APC-Cy7-conjugated anti-CD8α chain (SK1), and FITC-conjugated anti-CD8β chain (2ST8.5H7), in the presence of the live/dead fixable dead cell marker (Aqua LIVE/DEAD; Invitrogen), for 30 min at 4°. After washing with PBS, cells were fixed and permeabilized using the IntraPrep Fix/Perm Kit (Beckman Coulter) and incubated with antibodies specific for intracellular cytokines for 30 min at 4°: PE-conjugated anti-IL-2 (MQ1-17H12), PE-Cy7-conjugated anti-IFN-γ (B27), and APC-conjugated anti-TNF-α, all obtained from BD Biosciences. Cells were analysed using a BD FACSCanto flow cytometer (BD Biosciences) and data analysis was performed using FlowJo software. Human and NHP frozen PBMCs were thawed, rested overnight and distributed into 96-well plates (0·4 × 106 cells/well) coated with 50 μl anti-CD3 (OKT3, 1 μg/ml) and anti-CD28 (CD28.2; Beckman Coulter, 1 μg/ml) antibodies.

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