Brilliant violet 421 anti mouse ifn γ

To measure PD­L1 levels on the cell surface of tumour cells treated with vehicle, cisplatin, radiation, JQ1, IFN‐γ (20 ng/ml,²⁴ PeproTech), or the combinations for the different time periods were trypsinized and harvested for staining with streptavidin‐phycoerythrin (PE)‐conjugated anti‐human PD‐L1(329706, Biolegend). Tumour tissues were collected, cut into small pieces, digested with a mixture of collagenase, DNase and hyaluronidase for preparing single‐cell suspension and then treated with Cell Stimulation Cocktail (00‐4975‐03, eBioscience) as instructed. Cells were washed, re‐suspended in fluorescence‐activated cell sorting buffer at 4°C and then stained with fluorescent‐conjugated antibodies and appropriate isotype controls for multicolor flow cytometry. For cell surface staining, the following antibodies were used: fluorescein isothiocyanate (FITC) anti‐mouse CD3 (100204, Biolegend), PE/Cy7 anti‐mouse cluster of differentiation 4 (CD4) (100422, Biolegend), APC anti‐mouse CD8 (100712, Biolegend), Brilliant Violet 605 anti‐mouse NK1.1 (108739, Biolegend) and PE anti‐mouse PD‐L1(124308, Biolegend). The collected live single cells were fixed and treated with permeabilization buffer before intracellular staining with Brilliant Violet 421 anti‐mouse IFN‐γ (505830, Biolegend).

Anti-CCHFV NP and Gc rabbit polyclonal antibodies (pAb) were purchased from Integrated BioTherapeutics, Inc. (Gaithersburg, MD, USA). Anti-CCHFV Gn rabbit pAb was purchased from GenScript company (Nanjing, Jiangsu, China). The Sp2/0 mAb was supplied by our laboratory. Flow cytometry (FCM) antibodies, including APC/Cyanine7 anti-mouse CD3, FITC anti-mouse CD4, PE/Cyanine7 anti-mouse CD8, PE anti-mouse IL-4, and Brilliant Violet 421™ anti-mouse IFN-γ, were purchased from Biolegend, Inc. (San Diego, CA, USA). Other secondary antibodies, including FITC-conjugated and HRP-conjugated antibodies, were purchased from Proteintech Group, Inc. (Wuhan, Hubei, China). Purified CCHFV Gn and CCHFV NP were purchased from Abcam (Boston, MA, USA), and purified CCHFV Gc was purchased from The Native Antigen Company (Kidlington, Oxford, UK).