Apc conjugated anti ccr7 antibody

The expression of EPHB4 was detected via phycoerythrin (PE)‐conjugated EPHB4 antibody (R&D Systems, Inc., Minneapolis, MN, USA) staining. For the detection of EPHB4‐CAR expression, transduced T cells were stained using goat anti‐Ephrin B2 antibody (R&D Systems) and then stained using PE‐conjugated anti‐goat IgG antibody (R&D Systems). Allophycocyanin (APC)‐conjugated anti‐CD3 antibody, APC‐conjugated anti‐CD8a antibody, fluorescein isothiocyanate (FITC)‐conjugated CD4 antibody, FITC‐conjugated anti‐CD45RA antibody, and APC‐conjugated anti‐CCR7 antibody (all from BioLegend) were used for the characterisation of the CAR‐T cell phenotype. To determine the binding capacity of human Ephrin B2 to cynomolgus EPHB4, 293‐human EPHB4‐GFP and 293‐cynoEPHB4‐GFP cells were incubated with the recombinant human Ephrin B2‐Fc Chimera Protein (R&D Systems) for 20 min on ice and then stained using APC‐conjugated anti‐human IgG‐Fc antibody (BioLegend). Detailed antibody information is presented in Supplementary table 2. All flow cytometry data were acquired using BD FACS Accuri C6 Plus (BD Biosciences) and analysed using the FlowJo Software (Tree Star, Inc., Ashland, OR, USA).

Flow cytometry was carried out to analyze the abundance of the M1 marker CCR7, M2 marker CD206, and general macrophage marker F4/80. After 24 h of culture, BMDMs in the four groups were scraped, washed, blocked for 15 min with Blocking Buffer (Beyotime), and then stained for 1 h with an allophycocyanin (APC)-conjugated anti-CCR7 antibody (1:100, BioLegend, USA) or FITC-conjugated anti-CD206 antibody (1:100, BioLegend, USA). A PE-conjugated anti-F4/80 antibody (1:100, BioLegend, USA) was used to label all macrophages. The cells were analyzed using a BD flow cytometer with FlowJo software.