mitotracker green  (Thermo Fisher)

Journal: Journal of Traditional and Complementary Medicine

Article Title: Cinnamaldehyde inhibits the NLRP3 inflammasome and inflammatory response by reducing oxidative stress in Neisseria gonorrhoeae-infected macrophages

doi: 10.1016/j.jtcme.2025.06.003

Figure Lengend Snippet: Effect of CA on intracellular H 2 O 2 and mitochondrial damage in N. gonorrhoeae -infected macrophages. (A) J774A.1 macrophages were incubated with 20 μM CA or vehicle for 0.5 h before N. gonorrhoeae infection for an additional 4 h. The levels of intracellular H 2 O 2 were analysed by DCFH 2 -DA staining. (B – D) J774A.1 macrophages were incubated with 20 μM CA or vehicle for 0.5 h before N. gonorrhoeae infection for an additional 24 h. (B) The levels of mitochondrial ROS were analysed by MitoSOX staining. (C) Mitochondrial membrane integrity was analysed by MitoTracker Deep Red and MitoTracker Green staining. (D) The mitochondrial membrane potential was analysed by DiOC 2 (3) staining. The data are expressed as the mean ± SD of three separate experiments. ∗ p < 0.05 as indicated.

Article Snippet: MitoTracker Deep Red, MitoTracker Green, MitoSOX, 2′,7′-dichlorofluorescein diacetate (DCFH 2 -DA), DiOC 2 (3) and enzyme-linked immunosorbent assay (ELISA) kits for IL-1β, IL-6 and tumour necrosis factor-α (TNF-α) were obtained from Thermo Fisher Scientific (Waltham, MA).

Techniques: Infection, Incubation, Staining, Membrane

Journal: Materials Today Bio

Article Title: Extracellular biogenic nanoscale mitochondria reprogram the wound microenvironment via ROS scavenging independent of cellular uptake

doi: 10.1016/j.mtbio.2026.103023

Figure Lengend Snippet: Association between MSC-mt transfer and fibroblast survival (A) Flow cytometry analysis of the fluorescent signal of MitoTracker Deep Red-labeled MSC-mt in L929 cells after 16 h of co-culture with pharmacological inhibitors targeting different uptake pathways, showing that Dynasore selectively reduces mitochondrial transfer. (B) Flow cytometric analysis of apoptosis in L929 cells under the same inhibitor conditions as in (A), demonstrating increased cell viability despite reduced mitochondrial transfer upon inhibition of dynamin-dependent internalization. (C) Correlation analysis between MSC-mt fluorescent signal (MitoTracker fluorescence in A) and the proportion of viable cells (Annexin V-/PI- population in B), suggesting an inverse association between mitochondrial transfer and fibroblast survival. (D) Apoptosis levels between mt transfer+ (MitoTracker Deep Red + ) and mt transfer- (MitoTracker Deep Red − ) L929 cell populations, gated based on the subpopulations identified in A, showing consistently higher viability in the mt transfer − population. (E–F) Flow cytometry analysis of skin fibroblast apoptosis after 90 min and 180 min of co-culture with MitoTracker-labeled MSC-mt, showing MSC-mt alleviate apoptosis, with weakened protection observed for mt(R), and further enhancement of cytoprotection upon Dynasore treatment. (G) Flow cytometry analysis of MSC-mt fluorescent signal in skin fibroblasts at 90 min and 180 min post co-culture, showing that Dynasore significantly inhibits mitochondrial transfer at both time points. (H–I) Representative flow cytometry plots and quantification of apoptosis between mt transfer+ (MitoTracker Deep Red + ) and mt transfer- (MitoTracker Deep Red − ) skin fibroblasts populations, gated based on the subpopulations identified in G, showing that mt transfer + cells exhibit higher apoptotic burden compared with mt transfer − cells. All experiments were independently repeated three times (n = 3) and representative images are shown. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: For nanoparticle tracking analysis (NTA), iMSCs were first thawed and labeled with MitoTracker Green FM (CST, Cat# 9074) according to the manufacturer's instructions.

Techniques: Flow Cytometry, Labeling, Co-Culture Assay, Inhibition, Fluorescence

Journal: Materials Today Bio

Article Title: Extracellular biogenic nanoscale mitochondria reprogram the wound microenvironment via ROS scavenging independent of cellular uptake

doi: 10.1016/j.mtbio.2026.103023

Figure Lengend Snippet: Inhibition or physical blockade of mitochondrial entry attenuates H 2 O 2 -induced apoptosis and oxidative stress (A) Schematic diagram of the in vitro experimental design to assess the effect of mitochondrial internalization on cell fate under oxidative stress. (B) Flow cytometry analysis of apoptosis in L929 cells co-cultured with MitoTracker Deep Red-labeled MSC-mt for 24 h using Annexin V/PI staining. (C) Quantification of live cell percentages in each group based on Annexin V/PI staining results shown in B, showing enhanced cell survival when mitochondrial internalization is pharmacologically or physically restricted. (D) Analysis of MSC-mt fluorescence signal in L929 cells after 24 h of co-culture, showing effective reduction of mitochondrial internalization by Dynasore treatment and Transwell separation. (E) Comparison of apoptosis levels between mt transfer + (MitoTracker Deep Red + ) and mt transfer − (MitoTracker Deep Red − ) L929 cell populations, gated based on the subpopulations identified in D, indicating lower viability in mt transfer + population. (F–G) Representative flow cytometry plots and quantification of mitochondrial ROS levels in L929 cells after 24 h of co-culture with MSC-mt, showing more pronounced suppression of mtROS when mitochondrial transfer is restricted. (H) Measurement of H 2 O 2 concentration in the culture supernatant after 30 min of MSC-mt and L929 co-culture. (I) Measurement of H 2 O 2 concentration in the culture supernatant after 60 min of co-culture, showing effective extracellular ROS scavenging by MSC-mt. All experiments were independently repeated three times (n = 3) and representative images are shown. Data are presented as mean ± SEM. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ns, not significant. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: For nanoparticle tracking analysis (NTA), iMSCs were first thawed and labeled with MitoTracker Green FM (CST, Cat# 9074) according to the manufacturer's instructions.

Techniques: Inhibition, In Vitro, Flow Cytometry, Cell Culture, Labeling, Staining, Fluorescence, Co-Culture Assay, Comparison, Concentration Assay

Journal: Materials Today Bio

Article Title: Extracellular biogenic nanoscale mitochondria reprogram the wound microenvironment via ROS scavenging independent of cellular uptake

doi: 10.1016/j.mtbio.2026.103023

Figure Lengend Snippet: Immobilization of MSC-mt in thermosensitive hydrogel impairs their distribution and protective effects. (A) Photographs showing the thermosensitive properties of gel which remains liquid at 4 °C and transitions to a solid gel state at 37 °C. (B) Transmission electron microscopy images of MSC-mt after resuspension in hydrogel (final volume ratio: 50%). Mitochondria are indicated by red dashed outlines. Scale bar = 200 nm. (C) Quantification of free mitochondrial release after 3-h incubation in gels of different concentrations (10%, 20%, and 50%), with or without H 2 O 2 , showing a concentration-dependent restriction of mitochondrial release at higher gel densities. (D) Representative macroscopic images of mouse full-thickness skin wounds at the indicated post-wounding days following topical treatment with PBS (control), MSC-mt (mtH), 10% gel-embedded MSC-mt (10%gel + mtH) or 50% gel-embedded MSC-mt (50%gel + mtH). (E) Schematic diagrams illustrating wound areas at indicated time points. (F) Quantification of wound closure rates, showing accelerated healing in the 10%gel + mtH and mtH groups, but delayed closure in the 50%gel + mtH group (n = 8 mice per group). (G) Laser speckle blood flow imaging showing perfusion at the wound site on PWD7, indicating preserved microvascular perfusion in mtH and 10%gel + mtH groups, but reduced perfusion in the 50%gel + mtH group. (H) In vivo imaging of the spatial and temporal dynamics of MitoTracker-labeled MSC-mt at the wound site at 1 h, day 1, day 4, and day 7 after topical application, showing transient early retention with progressive signal loss over time. (I) Quantification of H 2 O 2 levels in the culture supernatant 1 h after co-treatment of L929 cells with H 2 O 2 and free or 50% gel-embedded MSC-mt, demonstrating impaired ROS neutralization when mitochondrial mobility is restricted by 50% gel. (J–K) Flow cytometry analysis and quantification of mitochondrial transfer by L929 cells after 24-h co-culture, showing reduced mitochondrial transfer in the 50% gel-embedded condition. (L–M) Representative plots and quantification of intracellular ROS levels using DCFH-DA staining after 24-h co-culture under oxidative stress, showing loss of ROS suppression when MSC-mt are immobilized within 50% gel. All in vitro experiments were independently repeated at least three times (n = 3∼4). For in vivo experiments, n = 6∼8 mice per group. Data are presented as mean ± SEM. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ns, not significant. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: For nanoparticle tracking analysis (NTA), iMSCs were first thawed and labeled with MitoTracker Green FM (CST, Cat# 9074) according to the manufacturer's instructions.

Techniques: Transmission Assay, Electron Microscopy, Incubation, Concentration Assay, Control, Imaging, In Vivo Imaging, Labeling, Neutralization, Flow Cytometry, Co-Culture Assay, Staining, In Vitro, In Vivo

Journal: Materials Today Bio

Article Title: Extracellular biogenic nanoscale mitochondria reprogram the wound microenvironment via ROS scavenging independent of cellular uptake

doi: 10.1016/j.mtbio.2026.103023

Figure Lengend Snippet: Association between MSC-mt transfer and fibroblast survival (A) Flow cytometry analysis of the fluorescent signal of MitoTracker Deep Red-labeled MSC-mt in L929 cells after 16 h of co-culture with pharmacological inhibitors targeting different uptake pathways, showing that Dynasore selectively reduces mitochondrial transfer. (B) Flow cytometric analysis of apoptosis in L929 cells under the same inhibitor conditions as in (A), demonstrating increased cell viability despite reduced mitochondrial transfer upon inhibition of dynamin-dependent internalization. (C) Correlation analysis between MSC-mt fluorescent signal (MitoTracker fluorescence in A) and the proportion of viable cells (Annexin V-/PI- population in B), suggesting an inverse association between mitochondrial transfer and fibroblast survival. (D) Apoptosis levels between mt transfer+ (MitoTracker Deep Red + ) and mt transfer- (MitoTracker Deep Red − ) L929 cell populations, gated based on the subpopulations identified in A, showing consistently higher viability in the mt transfer − population. (E–F) Flow cytometry analysis of skin fibroblast apoptosis after 90 min and 180 min of co-culture with MitoTracker-labeled MSC-mt, showing MSC-mt alleviate apoptosis, with weakened protection observed for mt(R), and further enhancement of cytoprotection upon Dynasore treatment. (G) Flow cytometry analysis of MSC-mt fluorescent signal in skin fibroblasts at 90 min and 180 min post co-culture, showing that Dynasore significantly inhibits mitochondrial transfer at both time points. (H–I) Representative flow cytometry plots and quantification of apoptosis between mt transfer+ (MitoTracker Deep Red + ) and mt transfer- (MitoTracker Deep Red − ) skin fibroblasts populations, gated based on the subpopulations identified in G, showing that mt transfer + cells exhibit higher apoptotic burden compared with mt transfer − cells. All experiments were independently repeated three times (n = 3) and representative images are shown. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: MSCs were pre-stained with MitoTracker Green (CST, Cat#9074) prior to mitochondrial isolation.

Techniques: Flow Cytometry, Labeling, Co-Culture Assay, Inhibition, Fluorescence

Journal: Materials Today Bio

Article Title: Extracellular biogenic nanoscale mitochondria reprogram the wound microenvironment via ROS scavenging independent of cellular uptake

doi: 10.1016/j.mtbio.2026.103023

Figure Lengend Snippet: Inhibition or physical blockade of mitochondrial entry attenuates H 2 O 2 -induced apoptosis and oxidative stress (A) Schematic diagram of the in vitro experimental design to assess the effect of mitochondrial internalization on cell fate under oxidative stress. (B) Flow cytometry analysis of apoptosis in L929 cells co-cultured with MitoTracker Deep Red-labeled MSC-mt for 24 h using Annexin V/PI staining. (C) Quantification of live cell percentages in each group based on Annexin V/PI staining results shown in B, showing enhanced cell survival when mitochondrial internalization is pharmacologically or physically restricted. (D) Analysis of MSC-mt fluorescence signal in L929 cells after 24 h of co-culture, showing effective reduction of mitochondrial internalization by Dynasore treatment and Transwell separation. (E) Comparison of apoptosis levels between mt transfer + (MitoTracker Deep Red + ) and mt transfer − (MitoTracker Deep Red − ) L929 cell populations, gated based on the subpopulations identified in D, indicating lower viability in mt transfer + population. (F–G) Representative flow cytometry plots and quantification of mitochondrial ROS levels in L929 cells after 24 h of co-culture with MSC-mt, showing more pronounced suppression of mtROS when mitochondrial transfer is restricted. (H) Measurement of H 2 O 2 concentration in the culture supernatant after 30 min of MSC-mt and L929 co-culture. (I) Measurement of H 2 O 2 concentration in the culture supernatant after 60 min of co-culture, showing effective extracellular ROS scavenging by MSC-mt. All experiments were independently repeated three times (n = 3) and representative images are shown. Data are presented as mean ± SEM. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ns, not significant. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: MSCs were pre-stained with MitoTracker Green (CST, Cat#9074) prior to mitochondrial isolation.

Techniques: Inhibition, In Vitro, Flow Cytometry, Cell Culture, Labeling, Staining, Fluorescence, Co-Culture Assay, Comparison, Concentration Assay

Journal: Materials Today Bio

Article Title: Extracellular biogenic nanoscale mitochondria reprogram the wound microenvironment via ROS scavenging independent of cellular uptake

doi: 10.1016/j.mtbio.2026.103023

Figure Lengend Snippet: Immobilization of MSC-mt in thermosensitive hydrogel impairs their distribution and protective effects. (A) Photographs showing the thermosensitive properties of gel which remains liquid at 4 °C and transitions to a solid gel state at 37 °C. (B) Transmission electron microscopy images of MSC-mt after resuspension in hydrogel (final volume ratio: 50%). Mitochondria are indicated by red dashed outlines. Scale bar = 200 nm. (C) Quantification of free mitochondrial release after 3-h incubation in gels of different concentrations (10%, 20%, and 50%), with or without H 2 O 2 , showing a concentration-dependent restriction of mitochondrial release at higher gel densities. (D) Representative macroscopic images of mouse full-thickness skin wounds at the indicated post-wounding days following topical treatment with PBS (control), MSC-mt (mtH), 10% gel-embedded MSC-mt (10%gel + mtH) or 50% gel-embedded MSC-mt (50%gel + mtH). (E) Schematic diagrams illustrating wound areas at indicated time points. (F) Quantification of wound closure rates, showing accelerated healing in the 10%gel + mtH and mtH groups, but delayed closure in the 50%gel + mtH group (n = 8 mice per group). (G) Laser speckle blood flow imaging showing perfusion at the wound site on PWD7, indicating preserved microvascular perfusion in mtH and 10%gel + mtH groups, but reduced perfusion in the 50%gel + mtH group. (H) In vivo imaging of the spatial and temporal dynamics of MitoTracker-labeled MSC-mt at the wound site at 1 h, day 1, day 4, and day 7 after topical application, showing transient early retention with progressive signal loss over time. (I) Quantification of H 2 O 2 levels in the culture supernatant 1 h after co-treatment of L929 cells with H 2 O 2 and free or 50% gel-embedded MSC-mt, demonstrating impaired ROS neutralization when mitochondrial mobility is restricted by 50% gel. (J–K) Flow cytometry analysis and quantification of mitochondrial transfer by L929 cells after 24-h co-culture, showing reduced mitochondrial transfer in the 50% gel-embedded condition. (L–M) Representative plots and quantification of intracellular ROS levels using DCFH-DA staining after 24-h co-culture under oxidative stress, showing loss of ROS suppression when MSC-mt are immobilized within 50% gel. All in vitro experiments were independently repeated at least three times (n = 3∼4). For in vivo experiments, n = 6∼8 mice per group. Data are presented as mean ± SEM. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ns, not significant. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: MSCs were pre-stained with MitoTracker Green (CST, Cat#9074) prior to mitochondrial isolation.

Techniques: Transmission Assay, Electron Microscopy, Incubation, Concentration Assay, Control, Imaging, In Vivo Imaging, Labeling, Neutralization, Flow Cytometry, Co-Culture Assay, Staining, In Vitro, In Vivo