Venous blood from antecubital veins were sampled in an EDTA-coated tube for flow cytometric analyses and a lithium heparin coated tube for serum CRP measurement. Flow cytometry was performed within 4 hours after blood sampling. We used an automated hematology analyzed Sysmex KX-21N (Sysmex Corporation, Kobe, Japan) to obtain the white blood cell count, which was used to calculate the volume of blood needed to obtain 5 × 10
5 white blood cells in each test tube. The red blood cells were lysed using 1% red blood cell lysis buffer (Nordic Biosite AB, Täby, Sweden) for 10 minutes at room temperature and in the dark. Cells were washed in an isotonic buffer (BD FACSFlow; BD Biosciences, Franklin Lakes, NJ, USA) and centrifuged for 5 minutes at 500
g. This process was repeated three times, after which the cells were resuspended in the isotonic buffer. We then added the following monoclonal antibodies: Allophycocyanine-CY7 CD11b, IgG1 κ, Clone ICRF44, Cat. No. 557754 (BD Biosciences); Phycoerythrin-CY7 CD14, IgG2a κ, Clone M5E2, Cat. No. 301814 (BioLegend, San Diego, CA, USA); and Phycoerythrin CD200, IgG1 κ, Clone 325516, Cat. No. FAB27241P (R&D Systems, Inc., Minneapolis, MN, USA). Flourochrome-matched negative isotypes were added to a separately prepared tube: Allophycocyanine-CY7 IgG1 κ, Clone MOPC-21, Cat. No. 400128 (BioLegend); Phycoerythrin-CY7 IgG2a κ, Clone MOPC-173, Cat. No. 400232 (BioLegend); and Phycoerythrin IgG1 κ, Clone MOPC-21, Cat. No. 555749 (BD Biosciences). Incubation was done at room temperature in the dark for 20 minutes, after which the cells were washed and resuspended in the isotonic FACSFlow buffer. Stained cells were analyzed using flow cytometry (BD FACSCanto II; BD Biosciences) with a sample size gated for 100,000 singlet leukocytes. Flow data were analyzed using Kaluza Analysis (Kaluza Analysis v. 1.5.20365.16139; Beckman Coulter, Inc., Pasadena, CA, USA) by two independent evaluators (YS and MKN) who were blinded to the conditions of the participants. We used cell height and area to gate singlets and size and granularity forward scatter [FSC]/side scatter [SSC] to gate monocytes, which was then used to investigate expression of the markers investigated (
Fig. 1). Nonspecific signaling was eliminated using corresponding negative isotype controls with a threshold of 1%.