Bromodeoxyuridine (brdu)

For S phase progression analyses with EdU incorporation, cells were treated with doxycycline (2 μg/ml) and 2 mM thymidine, 24 and 18 h before the addition of auxin 1000 μM, respectively. After the 4 h treatment with auxin, the cells were released, EdU was added 30 min before fixation and Click-iT reaction was performed with Click-iT™ EdU Cell Proliferation Kit for Imaging, Alexa Fluor™ 647 (Thermo Fisher Scientific), according to the manufacturer protocol.
For BrdU incorporation analysis in G2, cells were treated with doxycycline (2 μg/ml) and 2 mM thymidine, 24 and 18 h before the addition of auxin 1000 μM, respectively. After the 4 h treatment with auxin, the cells were released in RO3306, and BrdU (BioLegend) was added according with the manufacturer recommendation, and cells were fixed after 18 h.
For BrdU colocalisation with Ki-67 foci during replication progression, cells were treated with 2 mM thymidine for 18 h. After 18 h, the cells released and BrdU was added 1h before fixation according with the manufacturer.
For BrdU staining, cells were permeabilised for 10 min with 0.2% Triton-X, then 30 min with 2M HCL; the cells were then washed 3x 5 min wash with PBS. Thirty minutes of blocking at 37 °C with 1% BSA was followed, and finally 1 h incubation with Alexa Fluor® 647 anti-BrdU was done. The cells were mounted after washing with PBS 3x 5 min.

All animals were used between 8 and 16 weeks of age. Lifespan of cells was assessed using 50 μg/g body weight of BrdU (Biolegend, San Diego, CA, USA). BrdU staining was analyzed at different time points using the Phase‐Flow Alexa Fluor 647 BrdU Kit (Biolegend, San Diego, CA, USA) as well as surface antibodies to gate for different leukocyte populations. For subcutaneous IL‐4 application, osmotic pumps (Alzet 1007D, Durect Corporation, Cupertino, CA, USA) were implanted, filled either with IL‐4 (Miltenyi Biotec, Bergisch Gladbach, Nordrhein‐Westfalen, Germany) or vehicle only. IL‐4 was applied at 2.1 μg/kg/h over 5 days to achieve a concentration of approximately 200 pg/ml. Calculations were based on pharmacokinetic studies on recombinant IL‐4 in human patients²¹ and dose translation to mouse was done using the method published by Reagan‐Shaw.²² For the analysis of monocyte recruitment upon a mild sterile inflammatory stimulus, an intraperitoneal injection of 0.5 ml thioglycollate was performed, utilizing a modified protocol previously described by us.²³ Twelve hours after thioglycollate application, the peritoneal cavity was flushed with PBS supplemented with 3 mM EDTA and cells were analyzed by flow cytometry.