Percp conjugated anti cd3

We collected samples from age-, sex-, and basic disease-matched populations, excluding C. albicans BSI patients and healthy donors with hyperthyroidism and a history of insulin injection, because these conditions may affect the expression of GLUT3. The demographic information and clinical characteristics of the subjects are listed in Table 1. PBMCs from patients and healthy controls were extracted as described above. Isolated cells were stained with PerCP-conjugated anti-CD3, PE-conjugated anti-CD14 (BioLegend) and FITC-conjugated anti-GLUT3 (R&D Systems) to detect the expression of GLUT3 in monocytes and analyze using FlowJo 10.0 software.

For phenotypic characterization, 3 × 10⁵ cells were stained with fluorescein-isothiocyanate (FITC) anti-CD3, peridinin chlorophyll (PerCP)-conjugated anti-CD4, alexa fluor (AF) 700-conjugated CD8, brilliant violet (BV) 510-conjugated anti-CD45RA, allophycocyanin/Cyanin 7 (APC/Cy7)-conjugated anti-CD62L, anti-CD45RO-phycoerythrin (PE), and AF647-conjugated anti-CD197 (all BioLegend, London, Great Britian) monoclonal antibodies for 20 min at room temperature in the dark, washed with PBS (Lonza, Verviers, Belgium) with 0.1% human AB serum (C.C. pro, Oberdorla, Germany) and analyzed by multicolor flow cytometry (FACS Canto II, FACSDiva V8.1.2 software, BD Biosciences, Heidelberg, Germany). Gates were set based on the forward scatter versus side scatter properties of lymphocytes. At least 30,000 events were acquired in the CD3⁺ gate. For detailed gating strategy see Supplementary Figure S1B,C. For intracellular staining (ICS) peridinin chlorophyll (PerCP)-conjugated anti-CD3, alexa fluor (AF) 700-conjugated CD8, additional fluorescein isothiocyanate (FITC)-conjugated anti-IFN-γ and APC-conjugated TNF-α antibodies were used (both BioLegend). To determine Tregs and γδ T cells, alexa fluor (AF) 700-conjugated CD4, BV421-conjugated anti-CD25 (BioLegend), APC-conjugated anti-CD127, and anti-γδ TCR-PE/Cy7 antibodies were used (both BD Biosciences).

Specific T-cell phenotype and frequencies was assessed after treatment with 10 µM SnMP in an antigen-independent setting as well as before and after expansion of WT1-specific T cells. To distinguish between naïve T cells (T_N, CD62L⁺ CD45RA⁺), central memory T cells (T_CM, CD62L⁺ CD45RA⁻), effector memory T cells (T_EM, CD62L⁻ CD45RA⁻) or terminal differentiated effector memory T cells (T_EMRA, CD62L⁻ CD45RA⁺), cells were stained with peridinin chlorophyll (PerCP)-conjugated anti-CD3, allophycocyanin (APC)-conjugated anti-CD8, fluorescein isothiocyanate (FITC)-conjugated anti-CD19, APC/Cyanin 7 (Cy7)-conjugated anti-CD62L and anti-CD45RA-phycoerythrin (PE)/Cy7 (all BioLegend) and analyzed by multicolor flow cytometry (FACSCanto II, FACSDiva V8.1.2 software, BD Biosciences). Gates were set on the properties regarding light scatter of lymphocytes. At least 50,000 events were acquired in the CD3⁺ gate.