Mouse (C57BL/6; The Jackson Laboratory) CD4 T cells from spleen and lymph nodes were prepared (CD4 enrichment kit; STEMCELL Technologies) and CFSE labeled (Molecular Probes). Recipient mice were given vehicle or 30 mg/kg AM095 by gavage. 2 h later, 106 labeled CD4 T cells were adoptively transferred i.v. 16 h later, spleen or lymph nodes were collected and stained with anti-CD4-BV421 (clone GK1.5; BioLegend) and anti–CD8-APC (clone 53–6.7; eBioscience). The samples were analyzed with FACSAria flow cytometer (BD Biosciences). Data were analyzed with FlowJo software v 8.8.7 (Tree Star). Values are expressed as the percentage of CSFE+ CD4+ cells out of total CD4+ cells.
Spleen and lymph nodes were collected from above recipient mice and immediately submerged in OCT compound (Sakura Finetek). Tissues in OCT were quickly frozen using dry ice and then kept at −80°C for long-term storage. Sections of the lymph nodes were stained as above and imaged by confocal microscopy.
Spleen and lymph nodes were collected from above recipient mice and immediately submerged in OCT compound (Sakura Finetek). Tissues in OCT were quickly frozen using dry ice and then kept at −80°C for long-term storage. Sections of the lymph nodes were stained as above and imaged by confocal microscopy.