Apc anti human perforin

Pembrolizumab and nivolumab (anti-PD-1) were obtained from Merck and Bristol-Myers Squibb. The following antibodies were used for flow cytometry analyses: FITC anti-human CD4 (BioLegend Cat#391503), PE anti-human CD45 (Biolegend, clone HI30), APC anti-human CD56 (Biolegend, clone 5.1H11), APC/Cyanine7 anti-human CD8a (Biolegend, San Diego, CA, USA, clone RPA-T8), PE/Cyanine7 anti-human CD3 (Biolegend, clone HIT3a), PerCP/Cyanine5.5 anti-human CD314 (NKG2D) (Biolegend, clone 1D11), FITC anti-human CD6 (Biolegend, clone BL-CD6), APC anti-human CD16 (Biolegend, clone 3G8), Pacific Blue anti-human/mouse Ki67 (Biolegend, clone 16A8), FITC anti-human/mouse Granzyme B (Biolegend, clone GB11) and APC anti-human Perforin (Biolegend, clone B-D48).

Macrophages with or without rISG15 treatment for 12 h were stained with PE mouse anti-human CD163 (BD Bioscience, catalog no. 556018) and FITC mouse anti-human HLA-DR (BD Bioscience, catalog no. 560944). After gating the macrophages, a two-parameter density blot was used to distinguish M2 and M1 cells by creating a plot on CD163 (PE) and HLA-DR (FITC). For detecting the effectors of CD8⁺ cells, CD8⁺ cells were stained with PE anti-human CD8a (Biolegend, catalog no. 344705) prior to the intracellular staining with PE/Cyanine 7 anti-human IFN-γ (Biolegend, catalog no. 502527), or FITC anti-human/mouse Granzyme B (Biolegend, catalog no. 515403), or APC anti-human Perforin (Biolegend, catalog no. 308111). The intracellular staining permeabilization wash buffer (Biolegend, catalog no. 421002) is used to permeabilize CD8⁺ cells and following fixation with fixation buffer (Biolegend, catalog no. 420801). All data were collected using a cytoFLEX flow cytometer (BD Biosciences, USA) and analyzed using FlowJo software (BD Biosciences, USA).

UMCD6, a mouse anti-human monoclonal antibody that targets domain 1 of CD6, was created in our laboratory and was purified from mouse ascites and desalted by column chromatography using Protein G and dextran per manufacturer’s instructions (Thermo Fisher Scientific). Pembrolizumab and nivolumab (anti-PD-1) were obtained from Merck and Bristol-Myers Squibb. The following antibodies were used for flow cytometry analyses: FITC anti-human CD4 (BioLegend Cat#391503), PE anti-human CD45 (Biolegend, clone HI30), APC anti-human CD56 (Biolegend, clone 5.1H11), APC/Cyanine7 anti-human CD8a (Biolegend, San Diego, CA, USA, clone RPA-T8), PE/Cyanine7 anti-human CD3 (Biolegend, clone HIT3a), PerCP/Cyanine5.5 anti-human CD314 (NKG2D) (Biolegend, clone 1D11), FITC anti-human CD6 (Biolegend, clone BL-CD6), APC anti-human CD16 (Biolegend, clone 3G8), Pacific Blue anti-human/mouse Ki67 (Biolegend, clone 16A8), FITC anti-human/mouse Granzyme B (Biolegend, clone GB11) and APC anti-human Perforin (Biolegend, clone B-D48).