Anti cd3 fitc clone ucht1

Surface expression of meprin β and IL-6R was analyzed on human blood cells. Therefore human heparinized whole blood samples were blocked with Fc block (Human TruStain FcX, BioLegend) diluted 1:100 in FACS buffer (1% BSA [w/vol] in PBS) for 15 minutes and subsequently stained with either anti-meprin β (hEcto1, polyclonal rabbit against human meprin β ectodomain, Pineda) or the pre-immune serum as a negative control, diluted 1:500 in FACS buffer for 1 hour, followed by 30 minutes incubation with the secondary antibody, goat anti-rabbit Alexa Fluor 488 conjugate (Life Technologies), diluted 1:300. For detection of IL-6R and CD3, cells were stained with either anti-IL6R-APC (clone UV4, BioLegend) or the corresponding isotype control (mouse IgG1, κ BioLegend) or anti-CD3-FITC (clone UCHT1, Biolegend) diluted 1:100. All incubations were carried out at 4 °C. Lysis of erythrocytes was performed in RBC Lysis/Fixation Solution (BioLegend) subsequent to staining procedure. Labeled cells were analyzed utilizing a FACS Canto II flow cytometer (BD Biosciences) and data were evaluated with FlowJo (Tree Star) software. T cells were defined as CD3 + whereas granulocytes were gated by FSC/SSC as SSChigh.
All individuals underwent a written, informed-consent process approved by the ethics commission of the Medical Faculty of Kiel University (A 102/14).

The cells obtained from the digestion of the biopsies were washed and resuspended in PBS, then collected and transferred into 5 mL tubes for flow cytometry, and then incubated for 15 min in the dark with the Zombie Violet™ fixable viability stain (Biolegend, San Diego, CA, USA). The cells were then washed and resuspended in FACS buffer (PBS, 2% FBS, 2 mM FBS EDTA) to proceed with the labeling with antibodies (and their isotopic controls) for the recognition of surface markers. For immunophenotyping of T lymphocytes and TRM, the cells were labeled with anti-CD3 FITC clone UCHT1, anti-CD103 PE clone Ber-ACT8, anti-CD69 PerCP-Cy5.5 clone FN50 (Biolegend, San Diego, CA, USA), anti-CD8 PE-Cy7 clone HIT8a, anti-CD45 APC-Cy7 clone 2D1. In addition, the following antibodies were used for immunophenotyping NK and γδ T cells: anti-CD56 PE clone B159, anti-CD16 PECy7 clone 3G8, anti-Vδ2 APC clone B6 (Biolegend, San Diego, CA, USA). All the antibodies used, except for anti-CD69 and anti-Vδ2, were obtained from the BD Biosciences Company, (Franklin Lakes, NJ, USA). The samples were acquired using a BD FACSAria flow cytometer at least 50,000 viable cells were acquired for each sample. The gating strategy for the detection of T lymphocytes and TRM T cells is shown in supplemental Figure S1. The data obtained were analyzed using the FlowJo software (BD Biosciences).

For the analysis of monocyte populations in whole blood, 100 μl of EDTA blood was pipetted into Absolute Counting Tubes (Becton Dickinson) and incubated at room temperature for 20 min protected from the light with the following fluorochrome-conjugated monoclonal antibodies: anti-CD14-Alexa Fluor 647 (clone M5E2, Biolegend), anti-CD16-BV510 (clone 3G8, Biolegend), anti-CD66b-Alexa Fluor 700 (clone G10F5, Biolegend), anti-NKp46-FITC (clone 9E2, Biolegend), anti-CD11b-FITC (clone ICRF44, Biolegend), anti-CD3-FITC (clone UCHT1, Biolegend), anti-CD19-FITC (clone H1B19, Becton Dickinson), anti-HLA-DR-BUV395 (clone G46-6, Becton Dickinson), anti-CCR2-BV421 (clone 48,607, Becton Dickinson) and anti-CD45-BV786 (clone HI30, Becton Dickinson). After staining, the blood was incubated for 15 min at room temperature in the dark with 1-step fix/lyse solution (ThermoFisher). Stained fixed and lysed blood was stored at 4 °C in the dark before being run on an LSRFortessa™ with FACsDIVA software (Becton Dickinson) the next day. At least 5000 bead events were collected from each fully stained tube at the low acquiring speed and data was analysed using FlowJo software (TreeStar).