Anti cd19 percp cy 5.5 clone 1d3

To check the T-cell compartment in primary lymphoid organs due to TSLPR deficiency and anti-IL-7R administration, spleen and thymus were collected from all mice at day 36. They were weighed and single cell suspension was prepared in Hank’s balanced salt solution (HBSS; Invitrogen, Carlsbad, CA, USA) with 5% fetal bovine serum (FBS; Invitrogen) using a 70 μm cell strainer (Falcon, BD Bioscience, San Jose, CA, USA). Red blood cells in spleen were lyzed by 5 minute incubation in lysis buffer (Invitrogen). The reaction was stopped by addition of 10 mL cold PBS. Cells were counted, pelleted and resuspended in HBSS with 5% FBS and 1 μg Fc block (2.4G2; CD32/16, BD). Cells were incubated with monoclonal anti-CD4 APC/Cy7 (clone Gk1.5; BD), anti-CD8 Pacific Blue (clone 53–6.7; BD), anti-CD19 PerCP/Cy 5.5 (clone 1D3; BD), anti-CD44 PerCP/Cy 5.5 (clone IM7; eBioscience), and anti-CD62L APC (clone mel-14; BD). 10⁵ events were registered using FACS-LSRII (BD). Events were gated for viable lymphocytes based on forward and side scatter and specific staining using FlowJo software (Tree Star Inc, Ashland, OR, USA).

For cell culture, the following antibodies were used: anti-CD40 (rat IgG2a clone FGK45.5) from Miltenyi Biotec (Bergisch Gladbach, Germany) and anti-IgM F(ab´)₂ goat polyclonal antibody from Jackson ImmunoResearch (West Grove, PA, USA). For cell staining, the following antibodies were used: anti-CD19 FITC (clone 1D3/CD19), anti-GL-7 Pacific Blue (clone GL7), anti-Ki-67 Alexa 488 (clone 16A8), anti-pSTAT1 PE (clone A15158B), anti-pSTAT3 PE (clone 13A3-1), anti-pERK1/2 PE (clone 6B8B69), and anti-IL-6 PE (clone MP5-20F3) from BioLegend (San Diego, CA, USA), anti-CD19 PerCP-Cy5.5 (clone 1D3), and anti-CD21 APC (clone 7G6) from BD Biosciences (Mountain View, CA, USA), anti-CD138 PE (clone 30506), anti-CD23 PE-cyanine7 (clone B3B4) from Invitrogen (Carlsbad, CA, USA), anti-pSTAT5 PE (clone SRBCZX), anti-CD80 PE (clone 16-10A1), anti-CD86 PE (clone GL1), anti-BCL6 PE (clone BCL-DWN), and anti-IRF4 PE-Cy7 (clone 3E4) from eBioscience (San Diego, CA, USA), and anti-pAKT PE (clone REA359) from Miltenyi Biotec.