dcf da kit  (Yeasen Biotechnology)

Journal: ACS Nano

Article Title: Concurrent P‑Selectin Targeting Nanoparticle Orchestrates Tumor-Immune Dynamics for Advanced Immunochemotherapy

doi: 10.1021/acsnano.5c15120

Figure Lengend Snippet: Dual-cell targeting by FINAL promotes M1 polarization and suppresses tumor invasion in vitro . (A) Macrophage polarization in response to DTX-free or DTX-loaded NPs using RT-qPCR. n = 3 independent cell samples. (B) The influence of DTX-free or DTX-loaded NPs on the polarization of differentiated M2 macrophages through IHC staining. n = 3 independent cell samples, and the images were representative of 6 images. (C) The impact of DTX-free or DTX-loaded NPs on the invasion of M2 macrophages using a transwell coculture model. n = 3 independent cell samples, and the images were representative of 6 images. (D) The cROS level in MDA-MB-231 cells treated with DTX-free or DTX-loaded NPs for 24 h was assessed by utilizing 2′,7′-dichlorodihydrofluorescein diacetate (DCF-DA, 10 μM). Quantitative data are presented as fold change (FC), normalized to the control group. n = 3 independent cell samples. (E) DTX-free or DTX-loaded NPs to examine their effects on macrophage polarization in a transwell coculture model involving MDA-MB-231 cells and THP-1-M0, using RT-qPCR analysis. n = 3 independent cell samples. All DTX-free or DTX-loaded NP treatments were performed using an NP dosage standardized by DTX content, corresponding to 10 nM DTX. M0, nonactivated macrophage; M1, M1-macrophage; M2, M2-macrophage; MM231, MDA-MB-231; CM, conditioned medium; cROS, cellular ROS; CTL, control. Bar = 20 μm.

Article Snippet: Upon oxidation of DCF-DA to fluorescent 2′,7′-dichlorofluorescein (DCF), the emitted fluorescence signal was quantified using an Attune NxT flow cytometer (Thermo Fisher Scientific, USA) with the BL1 emission channel, employing a 488 nm excitation wavelength to assess intracellular ROS levels.

Techniques: In Vitro, Quantitative RT-PCR, Immunohistochemistry, Control

Journal: ACS Nano

Article Title: Concurrent P‑Selectin Targeting Nanoparticle Orchestrates Tumor-Immune Dynamics for Advanced Immunochemotherapy

doi: 10.1021/acsnano.5c15120

Figure Lengend Snippet: FINAL reshapes the TME and enhances therapeutic efficacy in vivo . (A) cROS levels of cancer cells (CD45 – /CD31 – ) within the tumor were detected using DCF-DA. n = 5 independent tumor samples. Quantitative data are presented as fold change (FC), normalized to the control group. (B) tSNE plots of TAMs (CD45 + /F4/80 + , gray) with an overlay of manually gated TNF-α + cells (red) are identified using flow cytometric biaxial gating strategies and pooled data. All samples = 60,000 events; individual samples = 12,000 events (from n = 6 independent tumor samples). (C) tSNE plots of tumor-infiltrating leukocytes (TILs, CD45 + , gray) with an overlay of manually gated M1 (CD86 + , red) and M2 (CD206 + , blue) are identified using flow cytometric biaxial gating strategies; and pooled data of M1/M2 in a stacked bar chart. All samples = 60,000 events; individual samples = 12,000 events (from n = 4 independent tumor samples). (D) Representative bioluminescence images of 4T1-Luc-bearing mice treated with Taxotere (10 mg kg –1 ), DTX@PNP (10 mg kg –1 ), and FINAL (10 and 20 mg kg –1 ) ( n = 6). (E) Representative images of the dissected tumors of the mice at 28-day post-tumor inoculation, and (F) the tumor volume change over the 28-day monitoring period ( n = 9 biologically independent mice). Arrows indicate the treatment days under the Q4d×4 regimen. (G) The survival of tumor-bearing mice was recorded. n = 5 independent tumor samples. (10), dosage at 10 mg kg –1 ; (20), dosage at 20 mg kg –1 ; MΦ, macrophage; M1, M1-macrophage; M2, M2-macrophage; CTL, control. * P < 0.05; ** P < 0.01; *** P < 0.001 were considered statistically significant versus the control. & P < 0.05; && P < 0.01; &&& P < 0.001 were considered statistically significant versus Taxotere(10). # P < 0.05; ## P < 0.01; ### P < 0.001 were considered statistically significant versus DTX@PNP(10). $ P < 0.05; $$ P < 0.01; $$$ P < 0.001 were considered statistically significant versus FINAL(10).

Article Snippet: Upon oxidation of DCF-DA to fluorescent 2′,7′-dichlorofluorescein (DCF), the emitted fluorescence signal was quantified using an Attune NxT flow cytometer (Thermo Fisher Scientific, USA) with the BL1 emission channel, employing a 488 nm excitation wavelength to assess intracellular ROS levels.

Techniques: Drug discovery, In Vivo, Control

Journal: Cancer Medicine

Article Title: Ubiquitin‐Specific Protease 45 Inhibits Lung Adenocarcinoma Ferroptosis by Regulating Ubiquitination and Stability of Glutathione Peroxidase 4

doi: 10.1002/cam4.71901

Figure Lengend Snippet: USP45 suppresses ferroptosis of lung adenocarcinoma cells induced by erastin. (A–C) Biochemical assays measuring levels of MDA, Fe 2+ , GSH, and GSSG in USP45‐depleted A549 and erastin‐treated USP45‐overexpressing HCC827 cells. USP45 deficiency amplified lipid peroxidation and oxidative imbalance, while USP45 overexpression reversed these effects. (D) ROS detection by DCF‐DA staining showing elevated ROS in USP45‐deficient A549 cells and reduced ROS in USP45‐overexpressing HCC827 cells in the presence of erastin. (E) Western blot analysis of ferroptosis markers ACSL4, COX2, and FTH1, showing increased ACSL4 and COX2 expression, reduced FTH1 expression in USP45‐deficient A549 cells, and the opposite pattern in USP45‐overexpressing HCC827 cells. One‐way ANOVA followed by Tukey's HSD tests was used to determine statistical significance. Abbreviation: Era, erastin.

Article Snippet: Approximately 1.0 × 10 6 cells were treated with 10 μM DCF‐DA (50101ES01, YEASEN) in PBS for 15 min in the dark.

Techniques: Amplification, Over Expression, Staining, Western Blot, Expressing

Journal: Journal of Integrative Plant Biology

Article Title: HSP101 ‐encoding NEO‐TETRAPLOID RICE FERTILITY GENE 1 regulates tapetum development through interaction with SAPK2 in polyploid rice

doi: 10.1111/jipb.70218

Figure Lengend Snippet: Reactive oxygen species (ROS) analysis in anthers of Huaduo1 (H1) and ntrf1 under exogenous abscisic acid (ABA) treatment (A) Nitroblue tetrazolium (NBT) staining of superoxide anion production in anthers of H1 and ntrf1‐1 at S7–S9 stages, with or without ABA treatment. Bars = 250 μm. (B) Analysis of ROS using 2’,7’‐dichlorodihydrofluorescein diacetate (H 2 DCF‐DA) in H1 and ntrf1‐1 anthers at S7–S9 stages, with or without ABA treatment. Bars = 250 μm. (C) 1% TTC staining of pollen grains and panicle observation. The scale bars for TTC and panicle morphology diagrams are 200 μm and 5 cm, respectively. (D) Relative quantification of ROS levels based on H 2 DCF‐DA fluorescence intensity in anthers at S7–S9 stages (means ± SD ; n = ~90). (E) Statistical analysis of mature pollen viability (means ± SD ; n = 25). (F) Statistical analysis of seed‐setting rates (means ± SD ; n = 10). (D – F) Different letters indicate significant differences ( P < 0.05; one‐way ANOVA, LSD test). (G) A working model of NTRF1‐SAPK2 interaction regulating pollen development in NTR. NTRF1 regulates ROS production by interacting with SAPK2. Normal ROS levels will promptly induce PCD in the tapetum, ensuring the formation of highly active pollen. In the ntrf1 mutant, ABA and ROS levels decrease, leading to pollen partial sterility.

Article Snippet: Additional staining was performed using H 2 DCF‐DA (MedChemExpress, 4091‐99‐0, USA).

Techniques: Staining, Quantitative Proteomics, Fluorescence, Mutagenesis, Sterility

Journal: International Journal of Molecular Sciences

Article Title: Subcytotoxic Exposure to Avobenzone and Ethylhexyl Salicylate Induces microRNA Modulation and Stress-Responsive PI3K/AKT and MAPK Signaling in Differentiated SH-SY5Y Cells

doi: 10.3390/ijms27031134

Figure Lengend Snippet: Effects of exposure to AVO and EHS on cell survival and ROS formation. Cells were differentiated with retinoic acid and treated for 48 h with UV filters. Cell viability ( a , b ) was evaluated by the reduction of resazurin to resorufin. ROS formation ( c , d ) was determined using the fluorescent probe H 2 DCF-DA. Data are expressed as fold increase versus the vehicle group and reported as mean ± SD of three independent experiments (( a ): * p < 0.05 vs. 0.05; ( b ): *** p < 0.001 vs. 0.05; One-way ANOVA, post hoc Tukey test).

Article Snippet: H 2 DCF-DA, eosin, retinoic acid, leupeptin, phenylmethylsulfonyl fluoride (PMSF), β-mercaptoethanol, sodium dodecyl sulfate (SDS), dimethyl sulfoxide (DMSO), Tris·HCl and β-actin antibody were obtained from Merck Life Science S.r.L. (Darmstadt, Germany).

Techniques: