Cd122 biotin

For flow cytometry, cells were isolated from the respective organs of mice. 1 * 10⁶ cells were washed twice with PBS in small flow cytometric tubes. Cells were incubated for 30 min with fixable Live/Dead 1:1000/PBS at 4°C. Afterwards cells were washed with PBS and incubated for 15 min with antibodies in FACS-buffer (2% BSA/PBS) and afterwards washed with PBS. For CD122 staining cells were additionally incubated with Streptavidin-PE. All antibodies [CD4-PacificBlue (Biolegend 100531), CD8-APC (Biolegend 100712), CD8-PerCPeFluor710 (Biolegend 100734), CD25-APC (Biolegend 102012), CD25-FITC (Biolegend 102006), GITR-PECy7, CD122-biotin (Biolegend 123206)] and Streptavidin-PE (eBioscience 12-4317-87) were used in 1:1000 dilution, except anti-humanCD2-FITC (Biolegend 300206) and anti-Foxp3-PE (eBioscience 12-5773-80) antibodies, which were both used in 1:200 dilution.

Isolated immune and inflammatory cells were incubated with primary antibodies for 20 min at 4 °C. After rising with 2 % FBS in PBS, cells were incubated with the secondary antibody Streptavidin PErCP (1:200, BD Biosciences) for 20 min at 4 °C (only for detection of CD122). Subsequent to rinsing, cells were exposed to Cytofix for 20 min at 4 °C and rinsed with BD Perm/Wash buffer (both reagents were purchased from BD Biosciences). Finally, we resuspended the cells using 2 % FBS in PBS. Flow cytometry was carried out using a FACSCalibur flow cytometer (BD Biosciences), with analysis being performed using CellQuest (BD Biosciences) for acquisition after exclusion of duplets and FlowJo 8.8.6 (Tree Star, Ashland, OR, USA). For each sample, we analyzed a total of 100,000 events (cells). We present the results as percentage of total cells analyzed, unless otherwise indicated.
We used the following primary antibodies (purchased from BD Biosciences, unless otherwise indicated), each at a dilution of 1:200: B220-FITC, MCHII-PE, CD4-APC, CD25-FITC, CD8-PE, CD122-biotin, CD11b-APC, CD45.2-FITC (BioLegend, USA), and NK1.1-PE.