Anti cd14 v500

Tumor and non-tumorous liver tissue from HCC patients was manually cut into small tissue fragments of 1-2 mm³. After processing, tissue fragments were placed in ultra-low adherence Nunclon™ Sphera™ 96-Well U-Shaped-Bottom Microplates in 100 μL of complete medium (RPMI-1640, 10% FBS, 1X GlutaMax, 1X Penicillin/Streptomycin, 1X Hepes, 1X non-essential amino acids, 1X Sodium Pyruvate, 0.01% β-mercaptoethanol). CD14⁺ cells were isolated from peripheral blood from the same patients. CD14⁺ cells were stained using the CellTrace Far Red (CTFR) Cell Proliferation kit (Biolegend) according to the manufacturer's protocol and 50,000 cells CTFR⁺ CD14⁺ cells were added to the wells containing tissue fragments. CTFR⁺ CD14⁺ cells were cultured alone as controls. After 3-7 days of co-culture cells and tissues were recovered, disaggregated mechanically, and filtered through 40 μm cell strainers (BD). Cell suspensions were stained with Zombie UV viability dye (Biolegend), anti-THY1 PE (1/50, Thermo Fisher), anti-CD45 PerCP-Cy5.5 (1/100, Miltenyi), anti-CD11b APC-Vio770 (1/100, Miltenyi) and anti-CD14 V500 (1/50, BD). Stained cells were analyzed using a FACS Symphony A5 instrument (BD).

Venous blood samples were collected in sodium heparin tubes and peripheral blood mononuclear cells isolated using Ficoll gradient centrifugation and cryopreserved in 10% Dimethyl Sulfoxide. Batched flow cytometry staining of frozen samples were performed including an even fraction of control and patient samples using the following antibodies for MAIT cell identification: Dead Cell Marker Aqua (Thermofisher, ratio 1:100), anti-CD14-V500 (BD Biosciences, clone: M5E2, ratio 1:100), anti-CD19-V500 (BD Biosciences, clone: HIB19, ratio: 1:100), anti-CD3-BV570 (Biolegend, clone:UCHT1, ratio: 1:50), anti-CD4-BUV615 (BD Biosciences, clone: SK3, ratio: 1:100), anti-CD161-BV650 (BD Biosciences, clone: DX12, ratio: 1:25), and anti-TCR Vα7.2-PE (Biolegend, clone: 3C10, ratio: 1:50). Cells were acquired using a BD FACSymphony A5. After compensation, MAIT cells were defined by first removing dead cells, B cells and monocytes to avoid background and unspecific binding of antibodies. Thereafter, total CD3-expressing cells were identified, CD4-positive cells excluded and MAIT cells defined as CD161 and TCR Vα7.2 double-expressing cells. A gating scheme to identify MAIT cells out of CD3⁺CD4^- cells and plots for CD161 and Vα7.2 in individuals with and without bacterial infection is presented in Fig 2.

A single flow cytometry panel was tested on all HIV-infected individuals and healthy control subjects as seen in Buggert et al. The antibodies used in the assay were: anti-CD3 APC-H7 (Clone SK7), anti-CD14 V500 (Clone M5E2), anti-CD19 V500 (Clone B43) (BD Bioscience); anti-CD45RO ECD (clone UCHL1) (Beckman Coulter); anti-CD4 BV650 (Clone OKT4), anti-PD-1 BV421 (clone EH12.2H7), anti-CD27 BV785 (clone O323), anti-CD28 PerCP-Cy5.5 (clone CD28.2), anti-CD38 APC (clone HIT2), anti-CD57 FITC (clone HCD57) (Biolegend); anti-HLA-DR PE-Cy7 (clone LN3) (eBioscience); anti-CD8 Qd565 (Clone 3B5) (Life Technologies) and anti-Tim-3 PE (clone 344823) (R&D Systems). LIVE/DEAD Aqua amine dye (Life Technologies) was used to discriminate dead cells and debris.