Brilliant violet

Characterization of lymphocyte subsets was performed using Multitest 6-color TBNK reagent containing CD3 FITC/CD16 PE + CD56 PE/CD45 PerCP-Cy5.5/CD4 PE-Cy7/CD19 APC/CD8 APC-Cy7 (BD Biosciences).
Analysis of PD-1 expression on T lymphocytes was performed using Brilliant Violet (BV510)-conjugated anti-PD-1 antibody (clone EH12.1 from BD Biosciences). Expression of PD-1 was measured as median fluorescence intensity (MFI) on CD3+ T lymphocyte population, derived from CD45+ lymphocyte population.
Expression of HLA-DR on monocytes was assessed using Brilliant Violet (BV421)-conjugated anti-HLA-DR (BD Biosciences) antibody (clone G46-6). HLA-DR expression was measured as MFI on the monocyte population obtained by gating on CD45 vs side scatter (SSC) population.
For characterization of Tregs subset, EDTA whole blood was stained with Human Regulatory T cell cocktail reagent [FITC anti-Human CD4/PE-Cy7 anti-Human CD25/Alexa Fluor 647 anti-Human CD127 (BD Biosciences)].
Stain-lyse-wash protocol was adopted for all flow cytometry assays mentioned above.

To assess the targeting of ME@C to the tumor region, we first administered intravenous injections of MCy5.5@C to 4T1 tumor–bearing BALB/c mice. The whole-body Cy5.5 spectral fluorescence intensity of the mice was measured at specific time intervals. In addition, the mice were humanely euthanized at various injection times, and tumor tissues were subjected to fluorescent imaging (Lumina XR, Caliper, USA).
To further confirm the in vivo targeting of M2-like TAMs, we injected MF@C (25 μg) intravenously into mice bearing 4T1 tumors. The tumors were harvested 24 hours after injection, and the uptake of ME@C was assessed using flow cytometry. Fluorescently labeled antibodies are as follows: 7-Aminoactinomycin D (7-ADD) [peridinin chlorophyll protein (PerCP)–Cy5.5, BD OptiBuild, 559925], CD45 (allophycocyanin-Cy7, BD OptiBuild, 557659), CD11b [phycoerythrin (PE), BD OptiBuild, 553311], F4/80 (Brilliant Violet, BD OptiBuild, 565411), and CD206 (Alexa Fluor, BD OptiBuild, 565250).