Anti cd69 mab

Frozen sections of the tonsils and spleens were acetone-fixed and stained for different markers with mAbs using standard protocols [25 ]. The following primary mAbs were used to identify lymphocytes, anti-CD8 (Abcam), anti-CD3 (Biolegend/ AbD Serotec) (to reveal CD8+ T cells) and anti-IgM (Life technologies) (to reveal B cells). The expression of CD69 and CD103 was identified using purified, flurophore-conjugated or biotin-conjugated anti-CD69 mAb (BioLegend) and anti-CD103 mAbs (BD Biosciences). The presence of IL-15 producing cells was determined using anti-IL-15 mAb (Abcam). The following secondary antibodies were used to reveal specific staining; affinity purified F(ab’)₂ fragments of AF647 or AF488 or Cy^TM3 conjugated donkey anti-mouse IgG and Cy^TM3 conjugated donkey anti-rabbit IgG (all obtained from Jackson ImmunoResearch). Control antibodies or secondary mAbs in the absence of primary mAbs were used to determine background fluorescent levels. Images were acquired using Delta Vision Personal (Olympus) or Zeiss LSM700 microscope and analysed Imaris software (Bitplane).

The expression of the cellular activation markers CD69 (early), CD25 (late) and HLA-DR (very late) were measured on freshly isolated PBMCs after 3 days of incubation at 37°C with varying concentrations of LA, PRO2000, tenofovir and PHA. Briefly, the cells were washed with PBS/FCS2% and incubated with PerCP-conjugated anti-CD4 mAb (BD Biosciences) together with PE-conjugated anti-CD25 mAb (BD Biosciences), anti-CD69 mAb (Biolegend) or anti-HLA-DR mAb (BD Biosciences) for 30 min. at 4°C. In parallel, the cells were stained with SimulTest control IgGγ1-FITC/IgGγ2a-PE (BD) for aspecific background staining. After washing, the cells were fixed with 1% paraformaldehyde solution and analyzed with the FACSCalibur and Cell Quest software (BD).