membrane potential assay kit  (Beyotime)

Journal: Genes & Diseases

Article Title: PDK1 elevation was induced by epigenetic modifications of KDM3A and METTL16 to mediate TKI resistance and cancer development

doi: 10.1016/j.gendis.2025.101947

Figure Lengend Snippet: PDK1 inhibitor JX06 and gefitinib synergistically induced cell apoptosis in gefitinib-resistant lung cancer cells. (A) The protein expression levels of PDK were reduced upon the treatment of PDK1 inhibitor JX06 in PC-9 and PC-9/G cells. (B) B2B and PC-9/G cells were treated with different concentrations of JX06 for 48 h. The cell viabilities were determined by CCK-8. (C – E) The synergy effect between JX06 and gefitinib was determined and analyzed with CompuSyn software. (F) The apoptosis rates were analyzed with flow cytometry after the combined treatment of gefitinib and JX06. (G) The TUNEL assay was performed with the indicated treatment in PC-9/G cells. (H) The cells treated as described were stained with the JC-1 probe and detected using a fluorescence microscope. Red fluorescence indicates the aggregation form of JC-1, showing increased mitochondrial membrane potential (ΔΨm). Green fluorescence indicates the monomeric form of JC-1, which indicates reduced mitochondrial membrane potential (ΔΨm). Data were statistically analyzed with Student’s t -test, and values were shown as mean ± standard deviation. ∗ P < 0.05 and ∗∗ P < 0.01.

Article Snippet: After different treatments for 48 h, the mitochondrial membrane potential of cells was detected with the enhanced mitochondrial membrane potential assay kit using JC-1 probe (Beyotime, Jiangsu, China), and the fluorescence was analyzed with a fluorescence microscope.

Techniques: Expressing, CCK-8 Assay, Software, Flow Cytometry, TUNEL Assay, Staining, Fluorescence, Microscopy, Membrane, Standard Deviation

Journal: Materials Today Bio

Article Title: Ultrasmall Prussian blue–integrated cryogel for enhanced ROS scavenging and immunomodulation via cGAS–STING inhibition in wound healing

doi: 10.1016/j.mtbio.2026.103056

Figure Lengend Snippet: Schematic representation of the synthesis process and therapeutic properties of Cryogel@USPB applied in wounds. (A) Fabrication process of Cryogel@USPB. (B) Cryogel@USPB promotes acute/chronic wounds through regulating macrophage polarization and restraining mitochondrial dysfunction via the cGAS-STING pathway.

Article Snippet: The Catalase Assay Kit and Mitochondrial Membrane Potential Assay Kit (JC-1) were from Beyotime Biotechnology Inc. (Shanghai, China).

Techniques:

Journal: Materials Today Bio

Article Title: Ultrasmall Prussian blue–integrated cryogel for enhanced ROS scavenging and immunomodulation via cGAS–STING inhibition in wound healing

doi: 10.1016/j.mtbio.2026.103056

Figure Lengend Snippet: The biocompatibility and antioxidant capability assessment of Cryogel@USPB. (A) Cell viability of NIH 3T3 and Raw264.7 cells after co-incubation with Cryogel@USPB at various concentrations (n = 3). (B) Representative fluorescent staining images of live (green)/dead (red) cells of Raw264.7 cells following co-incubation with Cryogel@USPB. (C) Determination of H 2 O 2 depletion capability of Cryogel@USPB (n = 3). (D) SOD-like activity and (E) POD-like activity of Cryogel@USPB (n = 3). (F) ·OH depletion capability and (G) •O 2 − depletion activity using EPR. (H) Representative fluorescent staining images of intracellular ROS in Raw264.7 cells following the treatment of H 2 O 2 and Cryogel@USPB. (I) TEM images of the mitochondria in RAW264.7 cells. (J) Fluorescent images of mitochondrial membrane potential in cells following different treatments. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The Catalase Assay Kit and Mitochondrial Membrane Potential Assay Kit (JC-1) were from Beyotime Biotechnology Inc. (Shanghai, China).

Techniques: Incubation, Staining, Activity Assay, Membrane

Journal: Cell Reports Medicine

Article Title: mRNA lipid-nanoparticle-mediated mitochondrial apoptosis augments adoptive T cell immunotherapy

doi: 10.1016/j.xcrm.2026.102706

Figure Lengend Snippet: mBH3@NPs preferentially induced mtApoptosis and immunogenic cell death in cancer cells (A) Flow cytometry analysis of cells positive for Annexin V and propidium iodide (PI) in cancer and non-cancer cell lines after treatment with PBS (control), NPs, naked Puma mRNA, and mPuma@NPs for 12 h ( n = 3). (B) Western blot analysis of mitochondrial apoptois pathway in CT-26 cells treated with control, NPs, mPuma@NPs, and mBim@NPs. Bax, Bcl-2, Bcl-x L , Mcl-1, caspase-3, cleaved caspase-3 (C-Cas3), caspase9, and cleaved caspase-9 (C-Cas9) proteins were detected. β-Actin was used as the loading control ( n = 3). (C) Confocal laser scanning microscopy (CLSM) images of the 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine (JC-1) probe in CT-26 cells after treatment with PBS (control), NPs, mPuma@NPs, and mBim@NPs for 12 h. Increased JC-1 monomer signal (green) and decreased JC-1 aggregate signal (red) indicate a decrease in mitochondrial membrane potential ( n = 3). Scale bars, 200 μm. (D) Flow cytometry analysis of cellular oxygen species (ROS) levels using 2,7-dichlorofluorescein diacetate (DCFH-DA) staining in CT-26 cells after 12 h incubation with PBS (control), NPs, naked mRNA, mPuma@NPs, and mBim@NPs ( n = 3). (E) CLSM images of CRT expression in B16-F10 and CT-26 cells after 12 h incubation with PBS (control), NPs, mPuma@NPs, and mBim@NPs ( n = 3). Scale bars, 20 μm. (F) Extracellular ATP and HMGB1 expression levels were analyzed by ELISA in B16-F10 cells after 12-h incubation with PBS (control), NPs, mPuma@NPs, and mBim@NPs ( n = 3). (G) Flow cytometry analysis and quantification of immune stimulation in BMDCs co-cultured with B16-F10 cells pretreated with PBS (control), NPs, mPuma@NPs, and mBim@NPs for 12 h, followed by 48-h co-culture ( n = 3). One-way ANOVA with Tukey’s multiple comparisons test was used for all statistical analyses. Data are presented as the mean ± SD. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; NS, not significant. See also .

Article Snippet: Mitochondrial Membrane Potential Assay Kit with JC-1 , Solarbio , Cat#M8650.

Techniques: Flow Cytometry, Control, Western Blot, Confocal Laser Scanning Microscopy, Membrane, Staining, Incubation, Expressing, Enzyme-linked Immunosorbent Assay, Cell Culture, Co-Culture Assay

Journal: Cell Reports Medicine

Article Title: mRNA lipid-nanoparticle-mediated mitochondrial apoptosis augments adoptive T cell immunotherapy

doi: 10.1016/j.xcrm.2026.102706

Figure Lengend Snippet: mBH3@NPs synergized T-cell-mediated cytotoxicity in vitro (A) Workflow of procedures for evaluating T-cell-mediated killing effects in target cancer cells. (B) Sensitivity of freshly isolated resting T cells and activated T cells to mBH3@NPs treatment ( n = 3). (C) Western blot analysis of altered protein expression in T cells after pre-activation for 3 days. Bax, Bcl-2, Bcl-x L , Mcl-1, caspase-3, and caspase-9 were detected. β-Actin was used as a loading control ( n = 3). (D) Quantification of B16-OVA tumor cell sensitivity to OT-1 T-cell-mediated killing following treatments of PBS, mPuma@NPs, and mBim@NPs at different E:T ratios (1:4, 1:2, 2:1, 4:1, and 10:1) by flow cytometry ( n = 3). (E) Flow cytometric quantification of B16-OVA tumor cell sensitivity to OT-1 T-cell-mediated killing following different dosages of mPuma@NPs (0.1, 0.25, 0.5, and 1 μg) at lower E:T ratios ( n = 3). (F) Flow cytometry histograms and quantification of Glut-1 OT-1 T cells co-cultured with B16-OVA cells treated with PBS, mPuma@NPs, and mBim@NPs. The co-culture was performed at an E:T ratio of 1:1 ( n = 3). (G) Glucose uptake capacity in OT-1 T cells was measured by flow cytometry using the 2-NBDG assay. (H) The cellular GSH:GSSG ratio was measured in OT-1 T cells after a 12-h co-culture with B16-OVA cells pre-treated with PBS, mPuma@NPs, and mBim@NPs. (I) Flow cytometry analysis of ROS levels of OT-1 T cells co-cultured with B16-OVA cells treated with PBS, mPuma@NPs, and mBim@NPs ( n = 3). (J) Mitochondrial membrane potential (Δψm) in OT-1 T cells was assessed by flow cytometry using the JC-1 probe after co-culture with B16-OVA cells treated with PBS, mPuma@NPs, and mBim@NPs. The Δψm is presented as the ratio of red (aggregates, high Δψm) to green (monomers, low Δψm) fluorescence intensity ( n = 3). (K) Flow cytometry histograms and quantification of PD-1 and LAG-3 expression of OT-I T cells co-cultured with B16-OVA cells treated with PBS, mPuma@NPs, and mBim@NPs ( n = 3). (L) Flow cytometry analysis of frequency of CD62L of OT-1 T cells co-cultured with B16-OVA cells treated with PBS, mPuma@NPs, and mBim@NPs ( n = 3). One-way ANOVA with Tukey’s multiple comparisons test was used for all statistical analyses. Data are presented as the mean ± SD. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; NS, not significant. See also .

Article Snippet: Mitochondrial Membrane Potential Assay Kit with JC-1 , Solarbio , Cat#M8650.

Techniques: In Vitro, Isolation, Western Blot, Expressing, Activation Assay, Control, Flow Cytometry, Cell Culture, Co-Culture Assay, Membrane, Fluorescence

Journal: Cell Reports Medicine

Article Title: A harmless-to-harmful switchable and spatiotemporally activated nano-CRISPR hierarchically amplifies ferroptosis in melanoma

doi: 10.1016/j.xcrm.2026.102718

Figure Lengend Snippet: The killing efficacy and ferroptosis efficacy of ARCHER in vitro (A) The Sanger sequencing result of the PCR amplicon of the targeted locus after treatment with CRISPR/Cas9-GPX4 by ARCHER. (B) The image of the PCR amplicon of the targeted locus by T7E1 assay. From top to bottom, the first gel strip is 549 bp in length. (C) Images of fluorescence of Calcein AM/PI in B16-F10 cells after different treatments. Scale bars represent 500 μm. (D) The fluorescence images of ROS generation in B16-F10 cells with different treatments. Scale bars represent 200 μm. (E) The representative images of FITC of positive cells percent and statistics by FCM, in which B16-F10 cells received different treatments and were stained with JC-1 by ordinary one-way ANOVA with Tukey’s multiple comparisons test (∗∗ p < 0.01; ns, not significant; n = 3; mean ± SD). (F) Statistics of mitochondrial membrane potential change of B16-F10 cells treated with different preparations by ordinary one-way ANOVA with Tukey’s multiple comparisons test (∗ p < 0.05, ∗∗∗ p < 0.001; ns, not significant; n = 3; mean ± SD). (G) The images of Bio-TEM of B16-F10 cells treated with ARCHER with/without laser. Scale bars represent 1 μm. (H) The confocal images of LPO in cells treated with different modalities and stained with C11 BOIDIPY. Scale bars represent 20 μm. (I–K) The analysis of LPO accumulation in B16-F10 cells receiving different nanoparticles treatment. The histogram for LPO (I) in single cells, quantitative analysis of FITC positive efficiency (J), and the MFI of FITC (K) of different nanoparticles on B16-F10 cells by ordinary one-way ANOVA with Tukey’s multiple comparisons test (∗∗∗ p < 0.001; ns, not significant; n = 3; mean ± SD). 1–7 refer to the treatment with PBS, RGD-R8-PEG-HA-ADH-Ce6Fe(III)Cl/PF33/CRISPR-Null, RGD-R8-PEG-HA-ADH-Ce6Fe(III)Cl/PF33/CRISPR-Null + laser, RGD-R8-PEG-HA-ADH-Ce6/PF33/CRISPR/Cas9-GPX4, RGD-R8-PEG-HA-ADH-Ce6/PF33/CRISPR/Cas9-GPX4 + laser, ARCHER, and ARCHER + laser.

Article Snippet: Mitochondrial membrane potential assay kit with JC-1 , Beyotime , Cat# C2006.

Techniques: In Vitro, Sequencing, Amplification, CRISPR, Stripping Membranes, Fluorescence, Staining, Membrane

Journal: Cell Reports Medicine

Article Title: mRNA lipid-nanoparticle-mediated mitochondrial apoptosis augments adoptive T cell immunotherapy

doi: 10.1016/j.xcrm.2026.102706

Figure Lengend Snippet: mBH3@NPs preferentially induced mtApoptosis and immunogenic cell death in cancer cells (A) Flow cytometry analysis of cells positive for Annexin V and propidium iodide (PI) in cancer and non-cancer cell lines after treatment with PBS (control), NPs, naked Puma mRNA, and mPuma@NPs for 12 h ( n = 3). (B) Western blot analysis of mitochondrial apoptois pathway in CT-26 cells treated with control, NPs, mPuma@NPs, and mBim@NPs. Bax, Bcl-2, Bcl-x L , Mcl-1, caspase-3, cleaved caspase-3 (C-Cas3), caspase9, and cleaved caspase-9 (C-Cas9) proteins were detected. β-Actin was used as the loading control ( n = 3). (C) Confocal laser scanning microscopy (CLSM) images of the 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine (JC-1) probe in CT-26 cells after treatment with PBS (control), NPs, mPuma@NPs, and mBim@NPs for 12 h. Increased JC-1 monomer signal (green) and decreased JC-1 aggregate signal (red) indicate a decrease in mitochondrial membrane potential ( n = 3). Scale bars, 200 μm. (D) Flow cytometry analysis of cellular oxygen species (ROS) levels using 2,7-dichlorofluorescein diacetate (DCFH-DA) staining in CT-26 cells after 12 h incubation with PBS (control), NPs, naked mRNA, mPuma@NPs, and mBim@NPs ( n = 3). (E) CLSM images of CRT expression in B16-F10 and CT-26 cells after 12 h incubation with PBS (control), NPs, mPuma@NPs, and mBim@NPs ( n = 3). Scale bars, 20 μm. (F) Extracellular ATP and HMGB1 expression levels were analyzed by ELISA in B16-F10 cells after 12-h incubation with PBS (control), NPs, mPuma@NPs, and mBim@NPs ( n = 3). (G) Flow cytometry analysis and quantification of immune stimulation in BMDCs co-cultured with B16-F10 cells pretreated with PBS (control), NPs, mPuma@NPs, and mBim@NPs for 12 h, followed by 48-h co-culture ( n = 3). One-way ANOVA with Tukey’s multiple comparisons test was used for all statistical analyses. Data are presented as the mean ± SD. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; NS, not significant. See also .

Article Snippet: CT-26 and B16-F10 cells (2×10 5 per well) were seeded in a 12-well plate, treated with PBS, NPs, mPuma@NPs, and mBim@NPs (1 μg mL −1 ) for 12 h. The level of ROS and mitochondrial membrane potential in cells were detected using the ROS Assay Kit and Mitochondrial membrane potential assay kit with JC-1 (Solarbio Science Technology Co., Ltd.), respectively.

Techniques: Flow Cytometry, Control, Western Blot, Confocal Laser Scanning Microscopy, Membrane, Staining, Incubation, Expressing, Enzyme-linked Immunosorbent Assay, Cell Culture, Co-Culture Assay

Journal: Cell Reports Medicine

Article Title: mRNA lipid-nanoparticle-mediated mitochondrial apoptosis augments adoptive T cell immunotherapy

doi: 10.1016/j.xcrm.2026.102706

Figure Lengend Snippet: mBH3@NPs synergized T-cell-mediated cytotoxicity in vitro (A) Workflow of procedures for evaluating T-cell-mediated killing effects in target cancer cells. (B) Sensitivity of freshly isolated resting T cells and activated T cells to mBH3@NPs treatment ( n = 3). (C) Western blot analysis of altered protein expression in T cells after pre-activation for 3 days. Bax, Bcl-2, Bcl-x L , Mcl-1, caspase-3, and caspase-9 were detected. β-Actin was used as a loading control ( n = 3). (D) Quantification of B16-OVA tumor cell sensitivity to OT-1 T-cell-mediated killing following treatments of PBS, mPuma@NPs, and mBim@NPs at different E:T ratios (1:4, 1:2, 2:1, 4:1, and 10:1) by flow cytometry ( n = 3). (E) Flow cytometric quantification of B16-OVA tumor cell sensitivity to OT-1 T-cell-mediated killing following different dosages of mPuma@NPs (0.1, 0.25, 0.5, and 1 μg) at lower E:T ratios ( n = 3). (F) Flow cytometry histograms and quantification of Glut-1 OT-1 T cells co-cultured with B16-OVA cells treated with PBS, mPuma@NPs, and mBim@NPs. The co-culture was performed at an E:T ratio of 1:1 ( n = 3). (G) Glucose uptake capacity in OT-1 T cells was measured by flow cytometry using the 2-NBDG assay. (H) The cellular GSH:GSSG ratio was measured in OT-1 T cells after a 12-h co-culture with B16-OVA cells pre-treated with PBS, mPuma@NPs, and mBim@NPs. (I) Flow cytometry analysis of ROS levels of OT-1 T cells co-cultured with B16-OVA cells treated with PBS, mPuma@NPs, and mBim@NPs ( n = 3). (J) Mitochondrial membrane potential (Δψm) in OT-1 T cells was assessed by flow cytometry using the JC-1 probe after co-culture with B16-OVA cells treated with PBS, mPuma@NPs, and mBim@NPs. The Δψm is presented as the ratio of red (aggregates, high Δψm) to green (monomers, low Δψm) fluorescence intensity ( n = 3). (K) Flow cytometry histograms and quantification of PD-1 and LAG-3 expression of OT-I T cells co-cultured with B16-OVA cells treated with PBS, mPuma@NPs, and mBim@NPs ( n = 3). (L) Flow cytometry analysis of frequency of CD62L of OT-1 T cells co-cultured with B16-OVA cells treated with PBS, mPuma@NPs, and mBim@NPs ( n = 3). One-way ANOVA with Tukey’s multiple comparisons test was used for all statistical analyses. Data are presented as the mean ± SD. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; NS, not significant. See also .

Article Snippet: CT-26 and B16-F10 cells (2×10 5 per well) were seeded in a 12-well plate, treated with PBS, NPs, mPuma@NPs, and mBim@NPs (1 μg mL −1 ) for 12 h. The level of ROS and mitochondrial membrane potential in cells were detected using the ROS Assay Kit and Mitochondrial membrane potential assay kit with JC-1 (Solarbio Science Technology Co., Ltd.), respectively.

Techniques: In Vitro, Isolation, Western Blot, Expressing, Activation Assay, Control, Flow Cytometry, Cell Culture, Co-Culture Assay, Membrane, Fluorescence