Pharm lysetm

For immunophenotype analysis, peripheral blood cells, bone marrow or spleen cells were resuspended in PBS/1% FBS after erythrocyte lysis (PharmLyse^TM, BD Pharmingen, San Diego, CA). Unless otherwise stated, the following antibodies were used: Sorting and analysis of LSK cells (Lin-Sca-1+cKit+) were performed as previously described [10 (link), 15 (link)]. Biotinylated antibodies against Gr-1 (RB6-8C5), B220 (RA3-6B2), CD19 (6D5), CD3 (145-2C11), CD4 (GK1.5), CD8 (53-6.7), TER119 and IL7Ra (A7R34) (all Biolegend, SanDiego, CA) were used for lineage staining. An APC-Cy7- or BV421-labeled streptavidin-antibody (BioLegend) was used for secondary staining together with an APC-anti-cKit (clone 2B8) and a PE-Cy7- or PE-anti-Sca-1 antibody (clone E13-161.7). Cells were analyzed using an BD-Fortessa, LSRII^TM or FACSCantoII^TM (Becton-Dickinson) cytometer. Analysis was performed using FlowJo^TM software (Treestar, Ashland, OR). Cell sorting was performed on a BD FACSAria™ II (Becton-Dickinson).

Anticoagulant (EDTA)-treated whole blood was collected to determine red blood cells, platelets, white blood cells, lymphocytes, neutrophils, basophils, and eosinophils (Green Cross Reference Lab, Kyungki, Korea).
Plasma was obtained by centrifugation, and IgG (Koma-Biotech, Seoul, Korea), IgA (Koma-Biotech), interleukin (IL)-2 (R&D systems, Minneapolis, MN, USA), IL-4 (BD Biosciences), tumor necrosis factor-α (TNF-α, BD Biosciences), and interferon-γ (INF-γ, R&D Systems) were measured according to the manufacturers’ protocols.
Red blood cells were lysed with Pharm-Lyse^TM (BD Biosciences), and lymphocytes were collected by centrifugation (200× g at 4 °C for 5 min). Collected lymphocytes were resuspended in PBS containing 1% fetal bovine serum and incubated with antibodies (against CD3, CD4, CD8, and CD45r) conjugated with florescent (BD Biosciences). Lymphocyte populations were measured with a FACSCalibur flow cytometer (BD Biosciences).

Flow cytometry immunophenotyping was performed on fresh bone marrow aspirate specimens using a standard staining and lyse/wash technique (PharmLyseTM, BD Biosciences, San Diego, CA, USA) as part of routine clinical work-up. Samples were examined with antibody panels designed to assess leukemic cells including: CD2, surface and cytoplasmic CD3, CD4, CD5, CD7, CD13, CD14, CD15, CD19, CD25, CD33, CD34, CD36, CD38, CD41, CD45, CD56, CD64, CD117, CD123, HLA-DR, MPO and TdT. An add-on panel for BPDCN was also performed in some cases consisting of CD2, CD4, CD7, CD34, CD38, CD45, CD56, CD64, CD123, CD303 and HLA-DR. For each analysis, a minimum of 200,000 events was acquired on CANTO II instruments (BD Biosciences), in which standardization was maintained using CS&T Beads with emphasis on comparable cross-platform performance. Data were analyzed using FCS Express (De novo Software, Glendale, CA, USA).
For the patterns and intensity assessed by flow cytometry, positivity for most markers was defined as expression in ≥20% of cells using the cutoff set by comparison to background fluorescence, with 20%–80% of cells being partially positive and >80% of cells being uniformly positive. A small subset was defined as expression in <20% of cells, but expression was deemed as clearly separated from the negative population.

Whole blood underwent red blood cell lysis (BD PharmLyseTM). Cells were washed and resuspended in PBS containing 0.5% bovine serum albumin (BSA) (Sigma-Aldrich) and 2 mM ethylenediamine tetra-acetate (EDTA) (Sigma-Aldrich). Fc receptors were blocked with a CD16/32 FcR Blocking Reagent (Miltenyi Biotec, 130-092-575) for 10 minutes at 4°C. Samples were then incubated with anti-CD45-VioBlue (Miltenyi Biotec, 130-110-802) [27 ], anti-CD11b-FITC (Miltenyi Biotec, 130-081-201) [28 ], anti-Ly6G-PE (Miltenyi Biotec, 130-107-913) [29 ], and anti-Ly6C-APC (eBioscience, 17-5932-82) [30 ] for 10 minutes at 4°C, according to the manufacturer’s protocol. Stained cells were washed in PBS containing 0.5% BSA and 2 mM EDTA. Samples were analyzed by flow cytometry (CytoFLEX S, Beckman Coulter). Leukocytes were identified based on typical light scatter properties, with further gating to define CD45⁺ leukocytes, CD45⁺CD11b⁺ myeloid cells, CD45⁺CD11b⁺Ly6G^-Ly6C^hi inflammatory monocytes, CD45⁺CD11b⁺Ly6G^-Ly6C^low reparative monocytes, CD45⁺CD11b⁺Ly6G^hiLy6C^hi neutrophils. Data were scaled to cells/mL of blood.

Angiogenic cells were enumerated by flow cytometer (LSRFortessa™ X20; BD Biosciences, USA) within 4 hours of blood draw. Whole blood collected in EDTA vacutainer (Becton, Dickinson and Company, UK) was incubated with anti-CD34 FITC, anti-VEGFR2 APC, anti-CD45 BV421, anti-CXCR4 APC-Cy7, and anti-CXCR7 PE (BioLegend, USA) in a Trucount™ tube (BD Biosciences, USA) with red blood cell lysis buffer (BD PharmLyse^TM, BD Biosciences, USA). Samples were analysed for 45 minutes or until 500,000 CD45⁺ events had been enumerated.
Flow cytometry files were analysed using FCS Express 7 (De Novo, California, USA). Counts of HPCs (CD34⁺) and EPCs (CD34⁺VEGFR2⁺) and subsequent cell surface expression of chemokine receptor 4 (CXCR4) and 7 (CXCR7) were converted to cells/mL using BD Trucount™, and adjusted for changes in blood volume [further methodology details available elsewhere (31 (link))]. Briefly, these phenotypes were chosen as low circulating CD34⁺ HPCs and CD34⁺VEGFR2⁺ counts are proven as risk factors to future adverse cardiovascular outcomes and death (25 (link), 27 (link)). Surface expression of CXCR4 and CXCR7 gives insight into how these cells respond to a stimuli such as exercise (31 (link)), characterising their ability to home to hypoxic environments, and may be a better predictor of mortality than HPCs and EPCs alone (27 (link)).