Journal: eLife

Article Title: Functional visualization of NK cell-mediated killing of metastatic single tumor cells

doi: 10.7554/eLife.76269

Figure Lengend Snippet: ( A ) NK cells derived from hyBRET-ERK-NLS mice were cultured with B16-R-GECO and observed under an epifluorescence microscope. Quantification of the FRET/CFP ratio in the NK cells that induced apoptosis (killer cells) and those that failed to induce apoptosis (non-killer cells) in the target cells. Data were pooled from six independent experiments and are shown as median ± SD; n=43 cells for killer cells and n=73 cells for non-killer cells. ( B ) Induction of apoptosis in the target cells by NK cells with or without ERK activation. Data are from six independent experiments. ( C ) NK cells were cultured with B16-R-GECO cells in the presence or absence of MEKi. Percentages of target cell death are shown. Data are pooled from three independent experiments and represented as means ± SDs. ( D ) NK cells derived from hyBRET-ERK-NLS mice are sorted by the expression of DNAM-1. The DNAM-1 + or DNAM-1 − NK cells were cultured with B16-R-GECO cells. Data were pooled from two independent experiments and are represented as median ± SD; n=37 cells for DNAM-1 + cells and n=27 cells for DNAM-1 − NK cells. ( E ) B16F10 cells or B16F10 Necl5 −/− Nectin2 −/− cells were stained with DNAM-1 Fc. The gray histogram is the background staining with a secondary Ab only. ( F ) The DNAM-1 + or DNAM-1 − NK cells derived from hyBRET-ERK-NLS mice were cultured with B16F10 Necl5 −/− Nectin2 −/− cells. Data were pooled from two independent experiments and are represented as median ± SD; n=38 cells for DNAM-1 + cells and n=41 cells for DNAM-1 − NK cells. ( G ) NK cells were cultured with B16F10 cells, or Necl5 −/− Nectin2 −/− B16F10 cell clones, A7, B7, and E7. Percentages of target cell death are shown. Data are pooled from three mice and represented as means ± SDs.

Article Snippet: The following antibodies were used for staining: BV510 or FITC anti-CD45 (30-F11), APC-Cy7 anti-CD3 (145–2C11), PerCP-Cy5.5 anti-NK1.1 (PK136), APC or PE-Cy7 anti-DNAM-1 (10E5), PE anti-F4/80 (BM8), APC anti-CD11b (M1/70), PE anti-NCR1 (29A1.4), PE anti-Ly-6G (1A8), PE anti-c-Kit (2B8), APC anti-CD49b (DX5), PE-Cy7 anti-CD200R3 (Ba13), PE anti-ICAM-1 (YN1/1.7.4), Alexa 488 anti-ICAM-2 (3C4), PE-anti PVR/Necl5 (TX56) (all from BioLegend, San Diego, CA), recombinant mouse DNAM-1 Fc chimera protein (R&D Systems), and Alexa Fluor 647 goat anti-mouse IgG (H + L) cross-adsorbed secondary antibody (Thermo Fisher Scientific).

Techniques: Derivative Assay, Cell Culture, Microscopy, Activation Assay, Expressing, Staining, Clone Assay

Journal: iScience

Article Title: Early TRAIL-engagement elicits potent multimodal targeting of melanoma by CD34 + progenitor cell-derived NK cells

doi: 10.1016/j.isci.2023.107078

Figure Lengend Snippet:

Article Snippet: To characterize receptor expression profile on NK cells, the following antibodies were used: anti-CD45 KO (J.33), anti-CD56 APCA750 (N901), anti-NKG2D PE (ON72), anti-NKp44 PE (Z231), anti-NKG2A PE (Z199), anti- KIR2DL1 KIR2DS1 PE (EB6.B) (all from Beckman Coulter), anti-DNAM-1 PE (REA1040) (Miltenyi Biotec B.V.), anti-NKp30 BV605 (p30-15), anti-NKp46 BV786 (9E2), anti-FASL BV605 (NOK-1), anti-TRAIL BV786 (RIK-2) (all from BD Biosciences) and 7-aminoactinomycin D (7-AAD; Sigma).

Techniques: Virus, Recombinant, Multiplexing, Activity Assay, Software

Journal: Cancers

Article Title: Chemotherapeutics Used for High-Risk Neuroblastoma Therapy Improve the Efficacy of Anti-GD2 Antibody Dinutuximab Beta in Preclinical Spheroid Models

doi: 10.3390/cancers15030904

Figure Lengend Snippet: Impact of chemotherapy on percentage of ( A ) cytotoxic NK cells of lymphocytes and NK-cell-specific activating receptors. ( B – E ) 5 × 10 6 PBMCs were treated for 24 h under cell culture conditions with either carboplatin (2 µg/mL, open circles), cisplatin (1 µg/mL, open triangles), etoposide (0.5 µg/mL, open squares), vincristine (0.05 µg/mL, open diamonds), or the cyclophosphamide metabolite (4-HPC, 1 µg/mL, open hexagons). After 72 h of culturing, cells were analyzed for NKp30, NKp44, NKp46, NKG2D, and CD226 expression, using flow cytometry. ( A ) Relative number of cytotoxic NK cells (CD3 − , CD56 dim ) in lymphocytes. ( B – E ) Geometric mean fluorescence intensity (gMFI) of respective activating receptor of cytotoxic NK cells after chemotherapy. Data represent at least four biological replicates. Means and SEM are indicated as black lines and error bars, respectively. For statistical analysis, repeated measures ANOVA with appropriate post hoc test was used; * p < 0.05 vs., ** p < 0.01 versus untreated control (medium).

Article Snippet: Incubation with the following antibodies in a total volume of 100 μL was conducted for 20 min at RT: CD3-VioGreen (REA613, 1:200), CD56-APC-Vio770 (REA196, 1:200), CD226-VioBlue (REA1040, 1:50); CD335 (NKp46)-Vio Bright B515 (REA808, 1:50), CD337 (NKp30)-PE (REA823, 1:75), CD336 (NKp44)-APC (REA1163, 1:75), and CD314 (NKG2D)-PE-Vio 770 (REA1228, 1:75), all from Miltenyi Biotec.

Techniques: Cell Culture, Expressing, Flow Cytometry, Fluorescence, Control

Journal: Scientific Reports

Article Title: The IgV domain of the poliovirus receptor alone is immunosuppressive and binds to its receptors with comparable affinity

doi: 10.1038/s41598-023-30999-w

Figure Lengend Snippet: Summary of vdPVR-Fc and PVR Fc binding kinetic parameters calculated from SPR sensorgrams.

Article Snippet: Binding kinetics of vdPVR-Fc, mutant vdPVR-Fc and PVR-Fc (786608; Biolegend) proteins to mTIGIT-Fc-his (771808; Biolegend), mCD226-his (50232-M08H; SinoBiological) and mCD96-his (788806; Biolegend) were derived from single-cycle kinetic analyses by surface plasmon resonance (SPR) using a Biacore T200 (Cytiva).

Techniques: Binding Assay