Brilliant violet 711

FOXP3‐eFluor 450 (eBioscience, Cat.# 48‐5773‐82, clone: FJK‐16s, 1:333), CD95 (Fas)‐Brilliant Violet 605 (BioLegend, Cat.# 152612, clone: SA367H8, 1:80), CD185 (CXCR5)‐Brilliant Violet 711 (BioLegend, Cat.# 145529, clone: L138D7, 1:40), CD4‐PerCP/Cyanine5.5 (BioLegend, Cat.# 100434, clone: GK1.5, 1:80), CD279 (PD‐1)‐PE (BioLegend, Cat.# 135206, clone: 29F.1A12, 1:20), CD25‐PE/Cy5 (BioLegend, Cat.# 102010, clone: PC61, 1:80), GL7‐eFluor 660 (eBioscience, Cat.# 50‐5902‐82, clone: GL7, 1:80), CD19‐APC/Cyanine7 (BioLegend, Cat.# 115530, clone: 6D5, 1:20), and LIVE/DEAD Fixable Yellow Dead Cell Stain (Thermo Fisher Scientific, Cat.# L34967, 1:7500).

The following antibodies were used for flow cytometry: CD11b (Brilliant Violet 605, BioLegend, cat. no. 101257), CD103 (APC/Cy7, BioLegend, cat. no. 121432), CD4 (APC, BioLegend, cat. no. 100411), CD45 (PerCP/Cy5.5, BioLegend, cat. no. 103132), CD8 (Brilliant Violet 711, BioLegend, cat. no. 100748), CXCR6 (PE, BioLegend, cat. no. 151103), Foxp3 (PerCP-Cy5.5, Biosciences, cat. no 563902), GFP (AF488, BioLegend, cat. no. 338008), and I-A/I-E (MHC-II, APC/Cy7, BioLegend, cat. no. 107628), CD103 (Brilliant Violet 605, BioLegend, cat. no. 121433), CD69 (Brilliant Violet 421, BD Biosciences, cat. no. 562920), CD45 (BV737, BD Biosciences, cat. no. 748371), and LIVE/DEAD fixable blue dead cell stain kit (Invitrogen, cat. no. L23105).

Cell surface labeling was performed using standard protocols. Intracellular labeling was performed using the True-Nuclear Transcription Factor Buffer Set according to the manufacturers’ instructions. The following anti-human mAbs were obtained from Biolegend: CD4-APC (OKT4), CD8α-PE (TuGh4), CD161-Alexa Fluor 647 (HP-3G10), CD69-PE (FN50), CD3-PE/Cy7, Brilliant Violet-711, or Alexa-700 (UCHT1), CD137-biotin (n4b4-1), CXCR3-Brilliant violet 421 (G025H7), CD83-biotin (HB15e) and TRAV1-2- PE (10C3). CD86-FITC (2331), CCR4-PECy7 (1G1) and CCR6-PE (11A9) mAbs were from BD Pharmingen. All these mAbs were used at 5 µg/ml. Biotinylated mAbs were revealed with streptavidin-PE, -Alexa Fluor 488, or -Brilliant violet 421 (2 µg/ml, Biolegend). The MR1-specific mAb clone 26.5 (mouse IgG2a) was provided by Ted Hansen, Marina Cella and Marco Colonna, Washington University School of Medicine, St. Louis (MO) (Lepore et al., 2014 (link)). Unlabeled MR1-specific mAbs were revealed with goat anti-mouse IgG2a-PE (2 µg/ml, Southern Biotech). Samples were acquired on LSR Fortessa flow cytometer (Becton Dickinson). Cell sorting experiments were performed using an Influx instrument (Becton Dickinson). Dead cells and doublets were excluded on the basis of forward scatter area and width, side scatter, and DAPI staining. All data were analyzed using FlowJo software (TreeStar).

Multicolor flow cytometry of peripheral blood cells was performed at Chungnam National University Hospital (CNUH). To exclude dead cells, single-cell suspensions were incubated for 20 min with a viability dye (LIVE/DEAD Fixable Aqua, Thermo Fisher). For neutrophil analysis, cells were stained with fluorochrome-conjugated antibodies, including anti-CD66b (Brilliant Violet 421; BD Biosciences), anti-CD56 (Brilliant Violet 510; BD Biosciences), anti-CD3, anti-CD19, anti-CD20 (Brilliant Violet 510; BioLegend), anti-PD-L1 (Brilliant Violet 711; BioLegend), anti-CD45 (Brilliant Violet 786; BioLegend), anti-CD10 (VioBright FITC; BioLegend), and anti-CD16 (APC-Cy7; BioLegend). For T-cell analysis, the cells were stained with fluorochrome-conjugated antibodies, including anti-CD14, anti-CD19 (Brilliant Violet 510; BD Biosciences), anti-CD4 (Brilliant Violet 876; BD Biosciences), anti-CD45RA (APC-R700; BD Biosciences), anti-CD3 (APC-Cy7; BD Biosciences), anti-CD8 (VioBright FITC; BioLegend), and anti-CCR7 (PerCP-Cy5.5; BioLegend). Flow cytometry was performed using a BD LSR Fortessa X-20 flow cytometer (BD Biosciences), and the data were analyzed using FlowJo software (Tree Star, Ashland, OR, USA). When gating subpopulations of cells, the distinction between positive and negative was based on Fluorescence Minus One (FMO) control.

For MDA-MB-231 and MCF10A cell lines: cells were enzymatically dissociated using trypsin-EDTA and stained as follows: cells were resuspended in ‘staining buffer’ (DPBS + 2% FBS) containing a human antibody against CD11b conjugated to the fluorophore Brilliant Violet 711 (BV711-CD11b; macrophage marker; Biolegend, 301344) at a 1:20–40 dilution. After a 30 min incubation on ice, cells were washed and resuspended in cold DPBS for analysis on the Fortessa. The background level of mEmerald fluorescence was set at 0.2% based on a fully stained co-culture control where macrophages were not transduced with mito-mEmerald. This gate was defined by FACS-isolating co-cultures of mito-RFP MDA-MB-231/mito-mEm macrophages and determining a gate that accurately isolated MDA-MB-231 cells containing macrophage mitochondria. We found that the cancer cell population with the highest mEm signal were cancer/macrophage fusions, and we therefore removed this population from downstream analysis. Setting the gate to 0.2% predominantly led to isolation of cancer cells with fragments of macrophage mitochondria, as visualized by microscopy.