Celltrace yellow

HMDMs were labeled on Day 5 prior to cytokine addition, and THP-1 cells were labeled on Day 0 prior to differentiation with PMA with CellTrace Yellow (C34567, Invitrogen) or CellTrace Far Red (C34564, Invitrogen) at 1:1000 in PBS for 30 min. LNCaP cells were labeled with CellTrace CFSE (C34554, Invitrogen) or CellTrace Violet (C34557, Invitrogen) at 1:1000 in PBS prior to apoptosis induction with cisplatin. Apoptotic LNCaP was added at a 5:1 apoptotic LNCaP cell:macrophage ratio in fresh media without cytokines. After 24 h, cells were harvested using trypLE Express (12,604–013, Gibco) and scraping. Cells were washed in PBS and resuspended in FACS buffer (PBS with 5% BSA and 2 mM EDTA). Efferocytosis quadrant gating was defined using a CellTrace Yellow- or CellTrace Far Red-labeled macrophage, unlabeled LNCaP cell control. Data were collected using an Attune NxT flow cytometer (Thermo Fisher Scientific) and analysis was performed using Kaluza (Beckman Coulter).

The following monoclonal antibodies (mAbs) and their isotype controls were used for flow cytometry: APC human PD-1, APC human PD-L1, Brilliant Violet human PD-L1, APC human CD86, APC human CD11b, PE human CD247 (CD3z), APC mouse CD11b; PE human EGFR from R&D Systems; APC mouse PD-L1 from Tonbo Biosciences. In addition, following reagents were used in flow cytometry: recombinant human PD-L1 Fc chimera Biotin protein (R&D Systems); APC Streptavidin (Tonbo Biosciences); Zombie NIR viability dye from BioLegend; CellTrace Violet, CellTrace CFSE, and CellTrace Yellow from Invitrogen.
For flow cytometry analysis, cells were resuspended in Cell Staining Buffer (BioLegend) containing Fc receptor blocker (Miltenyi Biotec) and incubated for 10 minutes at 4°C. Then the cells were stained in PBS containing 1:1000 diluted Zombie NIR viability dye (BioLegend) for 30 minutes at room temperature. Finally, the cells were stained with antibodies diluted in Cell Staining Buffer for 20 minutes at 4°C. Cells were washed twice with Cell Staining Buffer between the staining steps and once prior to data acquisition using SONY SA3800 or SONY iD7000 spectral flow cytometer. The flow cytometry data was analyzed using FlowJo software.

The flow cytometry-based cytotoxicity assay was performed using Cell Trace Violet (CTV)-labeled or Cell Trace Yellow (CTY)-labeled (Invitrogen) LMP2_426–434 pulsed as target cells as described before.⁴⁵ (link) Briefly, target cells were prepared by stimulating autologous PBMCs with the T cell mitogen phytohemagglutinin (PHA) (3 × 10⁶ PBMCs/mL were incubated in T cell medium with 20 μg/mL PHA) for 3 days at 37°C and 5% CO₂. Peptide-pulsed (or unpulsed) target cells were co-cultured with LMP2-specific T cells at various ratios for 4 h in CTL plus 10% horse serum. Unpulsed target cells alone were used as a control. After incubation, cells were harvested, stained using the LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Life Technologies) according to the manufacturer’s instructions, and viable target cells were quantified by flow cytometry using Flow Count Beads (Beckman Coulter, Brea, CA, USA). Cytotoxicity was calculated by comparing the percentage of viable target cells under test conditions relative to the control (100 − [target cell alive/target cell alone] × 100).

Prior to SAMD9/SAMD9L transfection 293 T cells were stained with either CellTrace™ Violet (for dye dilution experiment only) or CellTrace™ Yellow (for combination dye dilution and EdU incorporation cell cycle assays) (Invitrogen) according to the manufacturer’s protocol. In total, 72 h following transfection 293 T cells were collected, fixed with 4% paraformaldehyde, and analyzed by flow cytometry.

HMDMs were labeled on Day 5 prior to cytokine addition, and THP-1 cells were labeled on Day 0 prior to differentiation with PMA with CellTrace Yellow (C34567, Invitrogen) or CellTrace Far Red (C34564, Invitrogen) at 1:1000 in PBS for 30 min. LNCaP cells were labeled with CellTrace CFSE (C34554, Invitrogen) or CellTrace Violet (C34557, Invitrogen) at 1:1000 in PBS prior to apoptosis induction with cisplatin. Apoptotic LNCaP was added at a 5:1 apoptotic LNCaP cell:macrophage ratio in fresh media without cytokines. After 24 h, cells were harvested using trypLE Express (12,604–013, Gibco) and scraping. Cells were washed in PBS and resuspended in FACS buffer (PBS with 5% BSA and 2 mM EDTA). Efferocytosis quadrant gating was defined using a CellTrace Yellow- or CellTrace Far Red-labeled macrophage, unlabeled LNCaP cell control. Data were collected using an Attune NxT flow cytometer (Thermo Fisher Scientific) and analysis was performed using Kaluza (Beckman Coulter).

Prior to initiating the coculture, macrophages were labeled with CellTrace Violet (Invitrogen C34557) and target cells were labeled with 1:1000 diluted CellTrace CFSE (Invitrogen C34554) or CellTrace Yellow (Invitrogen C34567) for 30 minutes at 37 °C. In some conditions, macrophages were pre-treated with 2μM cytochalasin D (Cayman Chemical 11330), 10μg/mL anti-PD1 antibody (BioXCell SIM 0010) or 10μg/mL human IgG4 isotype control antibody (BioXCell CP147) prior to co-culture with target cells. Macrophages and target cells were cocultured at a 5:1 E:T ratio for 3 hours at 37 °C in ultra-low attachment 96-well plates (Corning 7007), unless otherwise indicated. Cells were stained for viability with Zombie NIR (BioLegend 423106) and in some experiments with APC-PD-1 antibody (BioLegend 329908). Data was acquired on SONY SA3800 spectral flow cytometer and analyzed using FlowJo software.