Anti hla abc apc clone w6 32

THP-1 cell line [32 (link)] was cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and antibiotics. Cells were washed with PBS pH 7.4 and incubated with either the protease-activated receptor 1 (PAR1) antagonist, FR171113 (10 μM, Tocris, Bristol, UK) [22 (link)], or PAR4 antagonist, (trans-Cinnamoyl)-YPGKF-NH₂ (tcY-NH₂, 10 μM, Tocris) [23 (link)] with or without CatG for 6 h at 37°C. DMSO served as a vehicle control. Subsequently, cells were washed in PBS and stained with anti-HLA-ABC-APC (clone W6/32; eBioscience, San Diego, California, USA) diluted in blocking buffer (1% FBS in PBS) for 30 min at 4°C. After two wash steps, cells were fixed in PBS containing 2% paraformaldehyde (PFA) and subsequently measured by a BD Canto II cytometer (Franklin Lakes, NJ, USA). Data were acquired and analyzed using FlowJo software (Tree Star, Inc., Ashland, OR, USA).

The following antibodies were used for cellular assays: anti-HLA-ABC-APC (clone W6/32, eBioscience), anti-CD3-BU737 (clone UCHT1, BD Bioscience), anti-CD3-PerCyP.5.5 (clone UCHT1, BioLegend), anti-CD19-BV510 (clone HIB19, BioLegend), anti-CD56-BU395 (clone NCAM16.2, BD Bioscience), anti-CD16-BV785 (clone 3G8, BioLegend), anti-KIR2DL1/S1/L3/S3/L5/S5-PE (clone HP-MA4, eBioscience), anti-KIR2DL1/S1-PE (clone 11PB6, Miltenyi), anti-KIR2DL1-FITC (clone REA284, Miltenyi), anti-KIR2DL3-APC (clone DX27, Miltenyi), anti-KIR3DL1/S1-BV421 (clone DX9, BioLegend), anti-NKG2A-PE-Cy7 (clone Z199, Beckman), anti-NKG2D-APC (clone 1D11, BioLegend), goat anti-human IgG(Fc) F(ab´)-PE (Life Technologies), anti-CD69-BV421 (clone FN50, BioLegend), anti-CD107a-BV421 (clone M4A3, BioLegend). For blocking assays we pre-incubated the samples with anti-HLA-C antibodies (clone 6A4, IgM, kindly provided by Prof. L. Moretta, Istituto Giannina, Genova, Italia) diluted 1/20 for 20 min at 37 °C.