Nbp1 00884

For FACS analysis, ESCs were trypsynized and purified from MEFs using the preplating method and EBs were disaggregated with Enzyme-Free Hanks’-based Cell Dissociation Buffer (Thermo Fisher Scientific). The obtained single cell suspension was incubated with 0.1% BSA and 1% FBS in PBS at room temperature for 30 min. Next, cells were incubated with primary antibodies on ice for 30 min. Antibodies against following antigens were used: IL-4Rα (diluted 1:50, Novus Biologicals NBP1-00884, Centennial, CO, USA), IL-13Rα1 (diluted 1:50, Novus Biologicals NBP1-61690), and VCAM1 (diluted 1:100, R&D Systems MAB6432, Minneapolis, MN, USA). Cells were washed with PBS once. Afterwards, cells were incubated with appropriate secondary antibody conjugated with APC-A (diluted 1:100, Thermo Fisher Scientific) at 4 °C for 30 min. Cells were fixed with 0.1% PFA at 4 °C for 10 min and analyzed with LSRFortessa cytometer (Becton Dickinson, Warsaw, Poland) and Diva 6.1 software.

Cells were fixed with 4% PFA and blocked for 1 h at RT with 10% protein block in PBS. Anti-IL-13Rα1 and anti-IL-4Rα (1:250, ab79277, Abcam, and 1:50, NBP1-00884, Novus Biologicals) antibodies were diluted in PBS with 1% protein block and were incubated overnight at 4 °C. Following washing, secondary antibody (donkey anti-rabbit IgG 555, 1:500) incubation was done for 1 h at RT. The specificity of the secondary antibody was tested by omitting the primary antibody. Counterstaining with DAPI was performed for 10 min. Pictures were taken using a LEICA DM4000 B LED microscope and LAS X software (Leica Microsystems).