Flow set

The flow cytometer was controlled daily using Flow‐Check (Beckman Coulter), and the stability of the compensations was examined daily using Flow‐Set (Beckman Coulter).
The absolute number of basophils and neutrophils was analysed. One hundred μl of whole blood was treated with the ImmunoPrep Reagent System (Beckman Coulter, USA) according to the manufacturer's instructions. Thereafter, the pretreated whole blood was mixed with 100 μl Flow‐Count beads (Beckman Coulter, USA), and the samples were analysed using flow cytometry, and the total number of leucocytes was counted.
Further, basophils were identified as CD203c+, CD193+ (Figure 1A,B) and neutrophils as CD15+, CD16+ (Figure 1C,D). Mean fluorescence intensity (MFI) was used to evaluate activation of basophils and neutrophils by expression of CD62L, CD11b and CD49d. In addition, basophil activation was also measured as MFI for CD203c and per cent positive CD63 basophils (%CD63+). A cut‐off for a negative test was set to a baseline CD63 expression of approximately 2·5%.

Fresh whole blood (100 µL) was labelled for surface and intracellular markers distributed in 3 flow cytometry panels: panel 1: CD3, CD8, CD4, CD45RA, CD27, CD28 and CD95, panel 2: CD3, CD8, CD4, CD45RA, CD27, CD28, Ki-67 and Sestrin-2 and panel 3: CD3, CD8, CD4, CD56, CD57, NKG2A, KLRG1 and DAP12 (see Table S4 for the specific clones and brand). All were direct stainings except for Sestrin-2. All stainings were performed according to the manufacturer’s recommendations, with Sestrin-2 expression being assessed after fixation and permeabilization (Biolegend FoxP3 Perm and Fix). After labelling, red blood cells were lysed using Versalyse lysing solution (Beckman Coulter, Inc., USA) according to the manufacturer’s recommendations. Surface and intracellular markers were analysed by flow cytometry using a Navios® flow cytometer (Beckman Coulter, Inc., USA) according to the manufacturer’s recommendations. Flow set and Flow-check fluorosphere (Beckman Coulter, Inc., USA) were used to calibrate our cytometer on days of experiment. Fluorescence minus one (FMO) controls were used to verify the absence of spillover after applying the compensation matrix and as gating controls. Additionally, isotype controls were used for Sestrin-2, Dap-12, Ki-67, NKG2A and CD95.