Lsrfortessa x 20 sorp flow cytometer

In this research, there were no more experiments performed. Immune staining and flow cytometry analyses have been performed in accordance with our previously published paper [7 (link)]. Briefly, peripheral blood mononuclear cells (PBMCs) were washed and suspended in flow cytometry staining buffer. Fc receptors (FcR) were first blocked using FcR Blocker (Miltenyi Biotec). Samples were then stained using particular antibodies that recognized cell surface or intracellular antigens. Samples were run on a BD LSRFortessa X-20 SORP flow cytometer, and data were collected using BD FACSDiva software (BD Biosciences), and then analyzed by FlowJo V10 software (FlowJo, Ashland, OR, USA).

Fresh whole blood staining was performed using 100 μl of blood for each sample, as previouly described [15 (link)]. Briefly, FcR Blocking Reagent (Miltenyi Biotec) was added first to block Fc receptors. Antibodies for myeloid markers (anti-human CD33-Fluorescein Isothiocyanate (FITC) (clone HIM3-4), CD11b-Allophycocyanin/Cyanine7 (APC-Cy7) (clone ICRF44), HLA-DR-phycoerythrin (PE) (clone G46-6), CD14-PE-Cy7 (clone M5E2), and CD15-APC (clone HI98)) (all from BD Biosciences, Oxford, UK) were added, and tubes were incubated at 4°C for 30 min. RBC lysis was performed using BD FACS lysing solution (BD Biosciences), as per the manufacturer's protocol. Cells were washed twice with PBS and resuspended in flow cytometry staining buffer (PBS + 1%FCS + 0.1%sodium azide).
Single-cell suspensions from tissues were washed with PBS, and the FcR Blocking Reagent (Miltenyi Biotec) was added, followed by staining with the aforementioned antibodies for myeloid markers. 7-AAD Viability Staining Solution (BioLegend, San Diego, USA) was used to gate live cells. Cells were incubated at 4°C for 30 min and then washed twice with PBS, followed by resuspension in flow cytometry staining buffer.
Data were acquired on a BD LSRFortessa X-20 SORP flow cytometer using BD FACSDiva software (BD Biosciences), and analyses were performed on FlowJo V10 software (FlowJo, Ashland, USA).