Iotest 3

Cells were thawed and 200 µl of cell suspension per well was transferred into 96-well plates. The cells were washed once with 200 µl Cell Staining Buffer (CSB from Biolegend) at 1400 rpm for 5 min. Cells were resuspended in 15 µl CSB supplemented with 15% FCR Block (Miltenyi) and incubated for 10 min at 4 °C. Fifty microliter of antibody mastermix for extracellular staining was then added to each well (Fig. S1). Samples were incubated for 30 min at 4 °C before being washed twice with 200 µl CSB. Finally, cells were filtered through a nylon mesh then were fixed in 200 µl of PBS supplemented with 0.5% IOTest3 (Beckman Coulter). As previously described, data were collected with a CytoFlex LX instrument (Beckman Coulter) and were subsequently analysed in Kaluza and CytoBank (Beckman Coulter) [9 (link)]. FlowSOM analysis was performed by applying an automated approach of clustering and visualization algorithms based on self-organizing maps [10 (link)].

Detached cells were scraped off with PBS, collected by centrifugation at 200 × G for 3 min. Cells (1 × 10⁶) were suspended in 100 µL staining buffer (BD Biosciences, 563794) and incubated with Fc block (BD Biosciences, 564220) for 10 min at 4 °C, then stained with anti-Human Ki67 (Affymetrix eBioscience, 25-5699-42) for 15 min. Cells were subsequently fixed in fixative solution IOTest 3 (Beckman Coulter, A07800) for 10 min and then washed twice with PBS. Cells were finally suspended in 500 µL sheath buffer and analyzed by BD FACSCANTO II (Biosciences, USA). A forward scatter (FSC) and side scatter (SSC) were used to select the whole population. The Ki67-positive cells were gated to calculate mean fluorescence intensity (MFI). The samples were run on a flow cytometer, collecting at least 20,000 events for each assay. Data analysis was conducted by FlowJo software (version 7.6.5, FlowJo LLC, Ashland, Oregon) (Supplementary Fig. S11).

For flow cytometric characterization of classical, intermediate, and non-classical monocytes directly in whole blood^{47 (link)}, freshly drawn heparinized blood (200 µl) was stained with CD14-PE, CD16-PC5, CD66b-APC, and CD45-PB for 15 min and treated with 2 ml of lysis solution (IOTest 3, Beckman Coulter) for 10 min at RT to lyse red blood cells. The remaining cells were pelleted for 5 min at 150 g, washed with PBS, resuspended in 500 µl PBS, and analyzed on a Gallios flow cytometer (Beckman Coulter) equipped with 405 nm, 488 nm, and 638 nm lasers. Data were analyzed using the Kaluza Software (Beckman Coulter). For gating, platelets and remaining red blood cells were excluded based on their CD45 expression, granulocytes were excluded based on their CD66b expression, and lymphocytes were excluded based on their expression of CD45 and CD14. Monocyte subsets were identified based on their expression of CD14 and CD16 as classical monocytes (CD14⁺⁺CD16⁻), intermediate monocytes (CD14⁺⁺CD16⁺), and non-classical monocytes (CD14⁺CD16⁺⁺)^{47 (link)} (n = 5). In addition to CD14 and CD16 surface expression, monocyte subsets were characterized via the exposure of chemokine receptors CCR2, CX₃CR1, CCR5 as summarized in Fig. 4c^{48 (link),49 (link)} (n = 3).