Live dead blue

First, cells were stained with a live/dead marker by incubating 3 × 10⁶ cells with 1:1000 v/v Live/dead blue (Life Technologies) in 100 µl PBS for 30 min at room temperature. The following washing and incubation steps were performed with fluorescence activated cell sorting (FACS) buffer (PBS [Life technologies] containing 1% FBS [Sigma-Aldrich], 0.09 % NaN₃ [Sigma-Aldrich], and 2 mM EDTA [Merck]). Blocking of Fc receptors was done before staining by incubating cells at 4 °C for 20 min with CD16/CD32 mAb either purified or labeled with PerCp/Cy5.5 (progenitor panel). After centrifugation cells were stained with all extracellular antibodies simultaneously at 4 °C for 20 min. The antibodies used are listed in Supplemental Table S1. We used spectral flow cytometry, which allows the use of fluorochromes with similar excitation and emission maxima in a single panel by measuring the entire emission spectrum of a fluorochrome and then unmixing the spectrum to identify individual fluorochromes, to simultaneously detect 28 cellular proteins After washing, cells were fixed with FoxP3 Fix/Perm buffer set (BioLegend) and stored over night at 4 °C in the dark. Intracellular staining was performed in FoxP3 Fix/Perm buffer at room temperature for 30 min. Samples were acquired on an AURORA spectral flow cytometer (Cytek Biosciences) and analyzed using OMIQ software (OMIQ).