Percp cy5.5 anti cd3

ILC1s were identified as Lin⁻CD34⁻CD94⁻TCRα/β⁻TCRγ/δ⁻CD1a⁻CD45⁺CD127⁺ CRTH2⁻CD117⁻ cells as described above. ILC2s were identified as Lin⁻CD34⁻CD94⁻TCRα/β^-TCRγ/δ^-CD1a⁻CD45⁺CD127⁺CRTH2⁺ cells. ILC3s were identified as Lin⁻CD34⁻CD94⁻TCRα/β^-TCRγ/δ^-CD1a⁻CD45⁺CD127⁺CRTH2⁻CD117⁺cells^{18 (link)}. pDCs were identified as Lin^-CD45⁺CD11c⁺CD11b⁻ cells using FITC anti-Lin, APC-H7 anti-CD45, PE anti-CD11b, and AF700 anti-CD11c (BD Bioscience, USA). mDCs were identified as Lin⁻CD45⁺CD11c⁺CD11b⁺ cells. T cells were identified as CD45⁺CD3⁺ cells using APC-H7 anti-CD45, Percp-cy5.5 anti-CD3 (BD Bioscience, USA). B cells were identified as CD45⁺CD19⁺ cells using APC-H7 anti-CD45 and APC-H7 anti-CD19 (BD Bioscience, USA). Natural killer (NK) cells were identified as CD45⁺CD56⁺ cells using APC-H7 anti-CD45, AF700 anti-CD56 (BD Bioscience, USA). Monocytes were identified as CD45⁺CD14⁺ cells using APC-H7 anti-CD45, FITC anti-CD14 (BD Bioscience, USA). For VCAM1 and MMP9 staining, PBMCs were separated from the 3^rd tertile group of STEMI patients and controls and stained with AF700 anti-VCAM1 and APC anti-MMP9 antibody (Biolegend, San Diego, USA). Viability was assessed by Aqua. After incubation with the respective antibodies for 30 minutes at 4 °C, cells were washed twice and subjected to flow cytometric analysis.

The thymuses were disaggregated by pressing through a 40 µm cell strainer (Fisher Scientific, Cat# 08-771-1) to obtain a single cell suspension. Thymocytes were stained with PerCP-Cy5.5-anti-CD3, APC-anti-CD4, and FITC-anti-CD8 antibodies (BD Bioscience, Franklin Lakes, NJ, USA) and analyzed by using FACS Aria (Becton, Dickinson and Company, Franklin Lakes, NJ, USA), using FACSDiva software v6.1.3 (BD Biosciences, Franklin Lakes, NJ, USA).

The isolated PBMCs were stained in duplicate with APC-H7-anti-CD4 (Becton Dickinson, San Diego, CA, USA), PerCP-Cy5.5-anti-CD3 (Becton Dickinson), BV510-anti-CCR4 (Becton Dickinson), PE-Cy7-anti-CCR6 (Becton Dickinson), BB515-anti-CCR10 (Becton Dickinson), and PE-CF594-anti-CXCR3 (Becton Dickinson) antibodies in the dark at 4 °C for 30 min. Negative controls were stained with isotype-matched control antibodies (APC-H7-anti-IgG1, PerCP-Cy5.5-anti-IgG1, BV510-anti-IgG1, PE-Cy7-anti-IgG1, BB515-anti-IgG2a, and PE-CF594-anti-IgG1). The frequencies of different T cells subsets were determined by flow cytometry analysis using FACSAria II (Beckton-Dickinson, San Diego, CA, USA) and analyzed by FlowJo software from Microsoft (v7.6.2, TreeStar, San Carlos, CA).

The PBMCs were stained in duplicate with APC-H7-anti-CD4 (Becton Dickinson, San Diego, CA, USA), PerCP-Cy5.5-anti-CD3 (Becton Dickinson), BV510-anti-CCR4 (Becton Dickinson), PE-Cy7-anti-CCR6 (Becton Dickinson), BB515-anti-CCR10 (Becton Dickinson), and PE-CF594-anti-CXCR3 (Becton Dickinson) antibodies in the dark at 4 °C for 30 min. Negative controls were stained with isotype-matched control antibodies (APC-H7-anti-IgG1, PerCP-Cy5.5-anti-IgG1, BV510-anti-IgG1, PE-Cy7-anti-IgG1, BB515-anti-IgG2a, PE-CF594-anti-IgG1). The frequencies of different T cell subsets were examined by flow cytometry analysis on a FACSAria II (Beckton Dickinson, San Diego, CA, USA) and analyzed by FlowJo software for Microsoft (v7.6.2, TreeStar, San Carlos, CA, USA).

To assess proliferation and further characterisation of the differentiation of NK cells, PBMC were permeabilised and stained with anti-Ki67-PE (eBioscience, Hatfield, UK), Granzyme-B-FITC and Perforin-Cy5.5/PerCP (Biolegend, London, UK) directly ex-vivo. For intracellular staining for IFNγ production; PBMC were incubated with rhIL12 and rhIL15 (10ng/ml) (R&D systems, Abingdon, UK), for 19 hours at 37°C. 1mM monensin (Sigma-Aldrich, Gillingham, UK) was added for the final 3 hours. Cells were then stained with anti-CD3-Cy5.5/PerCP or CD3/Pe-Cy7, CD16-APCy7, CD56-FITC, and subsequently fixed and permeabilised, followed by intracellular staining for IFNγ-v450 (BD Biosciences, Oxford, UK). Dead cells were excluded by fixable live dead stain.
For degranulation, PBMCs were incubated with K562 cells (5:1 E:T ratio) for 3 hours following overnight stimulation with a combination of 50ng/ml rhIL12 and rhIL18 (Miltenyi Biotech). Anti-CD107a-PE mAb (BD Biociences, Oxford, UK) was added at the time of stimulation with target-cells and monensin (1mM) added during the last 2 hours of incubation prior to staining and acquisition.