Anti hla dr percp cy5.5 clone g46 6

Each sample was aliquoted (300 µl) into one tube and stained with anti-IgE-PE (clone BE5, EXBIO Praha, Vestec, Czech Republic), anti-IgG-FITC (clone G18-145, BD), anti-HLA-DR- PerCP-Cy5.5 (clone G46-6, BD), anti-CD123-APC (clone AC145, Miltenyi Biotec; Bergisch, Gladbach, Germany), anti-CD16-PB (clone 3G8, Biolegend), anti-CD14-APC-H7 (clone MφP9, BD) and anti-CD45-krome orange (clone J.33, Beckman Coulter) for 15 min in the dark at RT. Then samples were incubated with 2 ml of FACS Lysing solution (BD) for 10 min in the dark at RT and centrifuged for 5 min at 540 g. The supernatant was discarded and the cell pellet was washed twice in 2 ml of PBS with centrifugation of 5 min at 540 g, resuspended in 0.5 ml of PBS, and stored at 4 °C before acquisition.

Six-parameter flow cytometric analysis was performed on whole blood, IELs, and T cells from LNs according to standard procedures (24 (link)) using a panel of monoclonal antibodies (mAbs) that were originally designed to detect human molecules but that we and others have shown to be cross-reactive with rhesus monkey (3 (link), 19 (link), 25 (link), 26 (link)). The antibodies used were as follows: anti-CD3-FITC (clone SP34), anti-CD4-APC-H7 (clone L200), anti-CD8-PE-Cy7 (clone RPA-T8), anti-CD69-PE (clone FN50), and anti-HLA-DR-PerCP-Cy5.5 (clone G46-6) (all from BD Pharmingen). Isotype antibody was used for negative control of CD69 and HLA-DR expression. Flow cytometric acquisition and analysis of samples were performed on at least 100,000 events on a BD Verse cytometer driven by the FACS Verse software (BD Biosciences). Analysis of the acquired data was performed using FlowJo software (TreeStar, Ashland, OR, USA). The CD4⁺ and CD8⁺ T cell percentages were based on the CD3⁺ T cells, and the activation markers on CD4⁺ and CD8⁺ T cells were based on CD4⁺CD3⁺ and CD8⁺CD3⁺ T cells, respectively.

After stimulation, samples were centrifuged at 500 x g for 3 min at 4°C. Cells were resuspended in 200 µl PBS (Gibco) supplemented with 2% FCS (Bodinco) and 2 mM EDTA (hereafter referred to as FACSbuffer) and washed. Cell pellets were incubated for 5 minutes with RBC lysis buffer (0.15 M NH₄Cl, 10 mM KHCO₃, 0.1 mM EDTA). If necessary, RBC lysis was repeated. After washing with PBS, cells were incubated for 30 minutes with Live/Dead stain (Invitrogen LIVE/DEAD^® Fixable Aqua Dead Cell Stain Kit) at 4°C. Cells were washed and incubated for 30 min at 4°C with the following fluorescent-labeled antibodies (Biolegend): anti-CD16 PE-Cy7 (clone 3G8), anti-CD14 APC-Cy7 (clone MΦP9), anti-HLA-DR PerCp-Cy5.5 (clone G46.6) from BD Pharmingen and anti-CD3 FITC (clone UCHT1), anti-CD19 FITC (clone HIB19), anti-CD56 FITC (clone HCD56) and anti-CD66b Pacific Blue (clone G10F5). Samples were washed and incubated with Fix/Perm solution (BD Cytofix/Cytoperm Fixation/Permeabilisation kit) at 4°C for 20 min. Thereafter, samples were incubated with α-IL-6 PE (clone MQ2-13A5) and α-TNF-α APC (clone Mab11) (Invitrogen) diluted in Perm buffer for 30 min at 4°C. Cells were washed and events were acquired at a BD FACSCanto™ II Flow Cytometer. All conditions were performed in duplicate. Analysis was performed using FlowJo version X (BD Biosciences).