Anti cd3 percp clone 145 2c11

Prediluted antibodies were prepared for staining of cell surface markers. For intracellular staining, cells were permeabilized in Foxp3 Fix/Perm solution (BioLegend) and incubated with anti-Foxp3 antibodies diluted in Foxp3 Perm buffer (BioLegend). The antibodies used: anti-CD3-PerCP (clone 145-2C11, BioLegend), anti-CD4-Pacific blue (clone RM4-5, BioLegend), anti-Foxp3-Alexa Fluor 488 (clone FJK-16s, Thermo Fisher Scientific), and anti-Nrp-1-APC (clone 3DS304M, Thermo Fisher Scientific). FACSCanto II (BD Biosciences) and FlowJo software (BD Biosciences) were used to collect and analyze the data.

The following antibodies and reagents were used to stain mouse cells: anti-CD4 Alexa Fluor 647 (clone RM4-5), anti-IFNγ Alexa Fluor 488 (clone XMG1.2), Streptavidin-PerCP (all BioLegend, San Diego, CA, USA), anti-CD25 biotin (clone 7D4, BD Pharmingen, Franklin Lakes, NJ, USA) anti-CD55 unconjugated (RIKO-3, Biolegend), anti-CD11b FITC (clone M1/70), anti-B220 Alexa Fluor 647 (clone RA3-6B2) (all BD Pharmingen), anti-CD3 PerCp (clone 145-2C11, BioLegend).
For staining of Pra1 a polyclonal antibody was raised in rabbits by immunization with purified recombinant Pra1. Aspf2-antiserum was generated by immunization of mice with purified recombinant Aspf2. Secondary polyclonal antibodies for staining of primary antibodies were goat anti-mouse-Ig FITC and donkey anti-rabbit-Ig PE (Jackson Immunoresearch, West Grove, PA, USA). Flow cytometry was performed on a FACSCalibur or LSR II flow cytometer using either CellQuest or DIVA software (BD Bioscience, Franklin Lakes, NJ, USA). We used FlowJo (TreeStar) to further analyze FACS data.

For flow cytometry, mesenteric lymph nodes (MLNs) were collected in HBSS supplemented with 5% FBS and 25 mM HEPES and strained through a 70 mM nylon membrane. Cells were incubated in HBSS supplemented with 3% FBS and 25 mM HEPES to avoid non-specific binding. Then a total of 1 × 10⁶ cells were stained with anti-CD4-PE (clone RM4-5), anti-CD8-APC (clone 53-6.6), and anti-CD3-PerCP (clone 145-2C11; all from Biolegend) for 1 h at 4°C. The cells were then fixed with 1% paraformaldehyde and analyzed by flow cytometry (BD Biosciences) at 10,000 total events/sample.