Cd3ε fitc

Cell number and viability of liver mononuclear cells were quantified using a TC20 Automated Cell Counter from Bio-Rad, then washed in staining buffer for characterization via flow cytometry. Blocking of Fc receptors was performed by incubation with TruStain FcX (BioLegend) for 10 minutes. Next, the cells were incubated with appropriate fluorochrome-conjugated antibodies for 30 minutes on ice (CD45-APC/Cy7, clone: 30-F11; CD3ε-FITC, clone: 145-2C11; CD4-BV786, clone: GK1.5; FoxP3-BV421, clone: MF-14 from BioLegend and RORyt-PE, clone: Q31-378 from BD Pharmingen). Cells were washed with staining buffer and then analyzed on a BD FACSCelesta flow cytometer. Data were analyzed with FlowJo v10 software.

H-2L^d/P1A and P91A tetramers (1 µM) were produced in our Institute^{53 (link)}. Antibodies used were: CD8α-PerCP (clone 53–6.7, Biolegend, 1 µg/mL); CD69-APC (clone H1.2F3, Biolegend, 1 µg/mL); CD3ε-FITC (clone 17A2, Biolegend, 1 µg/mL); CD45-FITC, CD45-Alexa700 (clone 30-F11, Biolegend, 1 µg/mL); Gr-1-APC, Gr-1-Bv421 (clone RB6-8C5, Biolegend, 1 µg/mL); Ly6C-PerCP (clone HK1.4, Biolegend, 1 µg/mL); Ly6G-PE, Ly6G-Bv510 (clone 1A8, Biolegend, 1 µg/mL); CD11b-BV421 (clone M1/70, Biolegend, 1 µg/mL); CD178 (FasL) (clone Kay-10, Biolegend, 2 µg/mL); CD31-PE/Cy7 (clone 390, Biolegend, 1 µg/mL); CD95-PE/Cy7 (Fas) (clone Jo2, Becton Dickinson, 1 µg/mL) and corresponding isotype controls. Viability dye efluor780 (eBiosciences, 1:500) was used. Annexin V-BV421 or Annexin V-APC (Biolegend, 1:50) was used to monitor apoptosis. P1A antibody (clone P1A102B3, 1 µg/mL) was produced by immunizing P1A-KO mice^{54 (link)} with a P1A peptide (CEEMGNPDGFSP) coupled to ovalbumin. Hybridomas were sub-cloned and further screened for production of anti-P1A antibodies. Clone P1A102B3 was selected and confirmed to produce an IgG1 recognizing P1A specifically and applicable for western blot, IHC, Flow cytometry and ELISA.

Antibodies against the following proteins were used: β-Actin (Merck, Madrid, Spain; monoclonal clone AC-15; #A5441), ADAM10 (Abcam, Cambridge, UK; rabbit polyclonal; #ab1997), N-Cadherin (Merck; rabbit monoclonal; #04–1126), Calreticulin (Thermo-Fisher, Barcelona, Spain; mouse monoclonal; #MA5–11723), CD19-PE-Cy5 (Biolegend, San Diego, CA, USA; clone 6D5; # 115509), CD3ε- FITC (Biolegend; clone 145-2C11; # 100305), CD314 (NKG2D)-PerCP-eFluor 710 (Thermo-Fisher; clone CX5; #46–5882-80), CD335 (NKp46)-APC (Biolegend; clone 29A1.4; #137607), CD45-PerCP-Cy5.5 (Biolegend; clone 30–711; Cat# 103131), CD8a-PE (Thermo-Fisher; clone 53.6.7; # 12–0081-82), F4/80-APC (eBioscience, San Diego, CA, USA; clone BM8; #17–4801-30), FLAG (Merck; monoclonal clone M2; #F1804), HIF1α (Novus, St Charles, MO, USA; monoclonal clone H1alpha67; #NB100–105), MLANA (Abcam, Cambridge, UK; mouse monoclonal; #ab200544), MITF (Millipore; mouse monoclonal; #MAB3747), Rae1δ (Thermo-Fisher; mouse monoclonal; #14–5756-81), HLA-ABC (Thermo-Fisher; mouse monoclonal; #MA5–11723), HLA-E (Thermo-Fisher; mouse monoclonal; #14–9953-80).