4-amino-5-methylamino-2",7"-difluorofluorescein diacetate (daf-fm da)  (Thermo Fisher)

Journal: Signal Transduction and Targeted Therapy

Article Title: Oleic acid restores the impaired antitumor immunity of γδ-T cells induced by palmitic acid

doi: 10.1038/s41392-025-02295-8

Figure Lengend Snippet: OA restores the reduced antitumor activity of Vγ9Vδ2-T cells induced by PA. a The apoptotic K562 cells were determined by flow cytometry after coculture with Vγ9Vδ2-T cells for 6 h ( n = 4). b The percentages of apoptotic tumor cells after coculture with Vγ9Vδ2-T cells for 6 h ( n = 4). c Diagram of the protocol in ( d – g ). Rag2 −/− γc −/− mice fed on the LFD, palm oil, olive oil or palm and olive oil HFDs for 30 days were subcutaneously (s.c.) injected with GFP + A549 tumor cells. Expanded BSA-, PA-, OA- or PA + OA-Vγ9Vδ2-T cells were intravenously (i.v.) transferred into the mice at the indicated time ( n = 6 mice per group). d Main components of control LFD, palm oil, olive oil, and palm and olive oil HFDs. e Tumor volumes were obtained at the indicated time. f On day 26 following GFP + A549 tumor cells inoculation, whole-body fluorescence images (left) and total radiant efficiency of fluorescence intensity (right) of the mice are shown after Vγ9Vδ2-T cells treatment. g Survival curves were acquired at the specified time. Quantitative data are shown as the mean ± SEM. ns not significant; * p < 0.05; ** p < 0.01; *** p < 0.001

Article Snippet: A NO fluorescence probe (DAF-FM DA, S0019S, Beyotime Biotechnology) was used to determine the intracellular NO release by a fluorescent microscope.

Techniques: Activity Assay, Flow Cytometry, Injection, Control, Fluorescence

Journal: Signal Transduction and Targeted Therapy

Article Title: Oleic acid restores the impaired antitumor immunity of γδ-T cells induced by palmitic acid

doi: 10.1038/s41392-025-02295-8

Figure Lengend Snippet: p STAT1-IRF1-iNOS pathway mediates IFNγ-induced pyroptosis in Vγ9Vδ2-T cells. a , b Vγ9Vδ2-T cells were cultured with BSA, PA, OA, or a mixture of PA and OA for 14 days. a p STAT1, IRF1, iNOS expression on Vγ9Vδ2-T cells, and NO level in their supernatants were examined ( n = 5). b The NO level in Vγ9Vδ2-T cells was quantified using DAF-FM DA probe under confocal microscopy. Representative confocal images and quantification of average fluorescence intensity are shown ( n = 5). The scale bar represents 20 µm. c , d Expanded BSA-, PA- or OA-Vγ9Vδ2-T cells were treated with or without anti-IFNγ Ab or rhIFNγ. c p STAT1, IRF1, and iNOS expression on Vγ9Vδ2-T cells and NO level in their supernatants were examined ( n = 5). d The NO level in Vγ9Vδ2-T cells was quantified using DAF-FM DA probe under confocal microscopy. Representative confocal images and quantification of average fluorescence intensity are shown ( n = 5). The scale bar represents 20 µm. The level of LDH release in the culture medium of BSA-, or PA-Vγ9Vδ2-T cells after fludarabine ( e ) or 1400 W ( f ) treatment was measured ( n = 5). g Representative immunoblot of GSDMD, cleaved GSDMD, caspase-1, and cleaved caspase-1 in BSA-, or PA-Vγ9Vδ2-T cells after fludarabine (left) or 1400 W (right) treatment. h – j Expanded BSA-, or OA- Vγ9Vδ2-T cells were treated with fludarabine or 1400 W, then rhIFNγ was used. h , i The level of LDH release in the culture medium of BSA-, or OA-Vγ9Vδ2-T cells was measured ( n = 6). j Representative immunoblot of GSDMD, cleaved GSDMD, caspase-1 and cleaved caspase-1 in BSA-, or OA-Vγ9Vδ2-T cells after fludarabine (left) or 1400 W (right) treatment. The percentages of apoptotic tumor cells after coculture with BSA-, or PA-Vγ9Vδ2-T cells with or without fludarabine ( k ) or 1400 W ( l ) treatment were examined ( n = 4). The percentages of apoptotic tumor cells after coculture with BSA-, or OA-Vγ9Vδ2-T cells in the presence of rhIFNγ with or without fludarabine ( m ) or 1400 W ( n ) treatment were examined ( n = 4). The data are shown as the mean ± SEM. ns not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001

Article Snippet: A NO fluorescence probe (DAF-FM DA, S0019S, Beyotime Biotechnology) was used to determine the intracellular NO release by a fluorescent microscope.

Techniques: Cell Culture, Expressing, Confocal Microscopy, Fluorescence, Western Blot

Journal: Signal Transduction and Targeted Therapy

Article Title: Oleic acid restores the impaired antitumor immunity of γδ-T cells induced by palmitic acid

doi: 10.1038/s41392-025-02295-8

Figure Lengend Snippet: Blockade of IFNγ and pyroptosis restores PA-impaired antitumor activity of Vγ9Vδ2-T cells in vivo. a Diagram of the protocol in ( b – f ). Rag2 −/− γc −/− mice fed on the LFD, palm oil, or olive oil HFDs for 30 days were subcutaneously (s.c.) injected with GFP + A549 tumor cells. Then anti-IFNγ Ab or rhIFNγ was intraperitoneally (i.p.) injected into mice. Expanded BSA-, PA-, or OA-Vγ9Vδ2-T cells were intravenously (i.v.) transferred into the mice at the indicated time ( n = 5 mice per group). b The level of IFNγ from mice serum collected on the 2 nd day after injection of anti-IFNγ Ab or rhIFNγ was measured. c Mouse weight was monitored for 2 months. d Tumor volumes were obtained at the indicated time. e On day 29 following GFP + A549 tumor cells inoculation, whole-body fluorescence images (upper) and total radiant efficiency of fluorescence intensity (lower) of the mice are shown after Vγ9Vδ2-T cell treatment. f Survival curves were obtained at the indicated time. g Diagram of the protocol in ( h – k ). Rag2 −/− γc −/− mice fed on the LFD, or palm oil HFD for 30 days were subcutaneously (s.c.) injected with GFP + A549 tumor cells. Then vehicle or DMF was intragastrically (i.g.) injected into mice. Expanded BSA-, or PA-Vγ9Vδ2-T cells were intravenously (i.v.) transferred into the mice at the indicated time ( n = 6 mice per group). h Mouse weight was monitored at the indicated time. i Tumor volumes were obtained at the indicated time. j On day 29 following GFP + A549 tumor cells inoculation, whole-body fluorescence images (upper) and total radiant efficiency of fluorescence intensity (lower) of the mice were assessed after Vγ9Vδ2-T cell treatment. k Survival curves were acquired at the specified time. l Schematic overview of the effect of PA and OA on the antitumor activity and the mechanism of IFNγ-induced pyroptosis in Vγ9Vδ2-T cells. The schematic was made using Biorender and included a publication license (Zhang (2025) https://BioRender.com/h90t444 ). Quantitative data are shown as the mean ± SEM. ns not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001

Article Snippet: A NO fluorescence probe (DAF-FM DA, S0019S, Beyotime Biotechnology) was used to determine the intracellular NO release by a fluorescent microscope.

Techniques: Activity Assay, In Vivo, Injection, Fluorescence