red-c12  (Thermo Fisher)

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: MTFR2-Mediated Fission Drives Fatty Acid and Mitochondrial Co-Transfer from Hepatic Stellate Cells to Tumor Cells Fueling Oncogenesis.

doi: 10.1002/advs.202416419

Figure Lengend Snippet: Figure 3. Activated HSCs enhance fatty acid (FA) synthesis and transfer to cancer cells, promoting proliferation. A) Western blot showing the ACC1 and CPT1A level of Huh7 cells and Huh7 cells cocultured with aHSCs (n = 3). B) Schematic of the experimental setup for FA chase pulse assay. HSCs were incubated in complete media (CM) with red fluorescent FA (Red C12) for 16 h (“pulse”). Following this, HSCs were cultured for an additional 72 h in CM or tumor-conditioned media (TCM) without the labeled FA (“chase”). Mitochondria and lipid drops (LDs) were stained before imaging. C,D) FA localization was assayed as described in (A) and chased in CM or TCM. C) LDs were labeled using BODIPY 493/503 (gray) and mitochondria were labeled using MitoTracker Far Red (green). Scale bar = 25 μm. D) Relative cellular localization of Red C12 was quantified by Pearson’s coefficient analysis

Article Snippet: For FA transfer assays, HSCs were preloaded with 1 × 10−3 m Red C12 (BODIPY 558/568 C12, MCE, Catalog No. HY-138226) in CM for 16 h, then rigorously washed three times with CM and equilibrated for 1 h to remove extracellular dye aggregates.

Techniques: Western Blot, Incubation, Cell Culture, Labeling, Staining, Imaging

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: MTFR2-Mediated Fission Drives Fatty Acid and Mitochondrial Co-Transfer from Hepatic Stellate Cells to Tumor Cells Fueling Oncogenesis.

doi: 10.1002/advs.202416419

Figure Lengend Snippet: Figure 5. Complementary roles of FAs transfer and mitochondrial transport in supporting tumor cell survival. A) Representative confocal images of Huh7 cells cocultured with aHSCs under L-778123 and NTL treatment. Maximum intensity projections (MIP) of confocal Z-stacks showing Huh7 cells (CellTrace green, green) cocultured with aHSCs pre-labeled with Red C12 fluorescent FA (red) (Upper panels). Corresponding 3D surface renderings (Imaris) of the boxed regions (Lower panels). Scale bars: 15 μm. B) Graph showing the fluorescence intensity of FAs in CellTrace green labeled Huh7 cells (Data are represented as mean ± SD, n = 6, at least 50 recipient cells analyzed per replicate, one-way ANOVA was performed). C) Flow cytometry analysis of fluorescence intensity of FAs in FITC-positive Huh7 cells. D) Confocal images showing the transportation of mitochondria from aHSCs to Huh7 cells. (Upper panels) MIP: Confocal Z-stacks of cocultured MitoTracker-labeled mitochondria in aHSCs (red) and CellTrace green-labeled Huh7 cells (green), with ActinTracker (cyan) highlighting tunneling nanotubes (TNTs). (Lower panels) 3D surface reconstruction (Imaris). Scale bars: 15 μm. E) Quantification of fluorescence intensity from Figure D, showing the uptake of HSC-derived mitochondria by Huh7 cells (Data are represented as mean ± SD, n = 6, at least 50 recipient cells analyzed per replicate, one-way ANOVA was performed). F) Fluorescence histograms of Huh7 cells (labeled

Article Snippet: For FA transfer assays, HSCs were preloaded with 1 × 10−3 m Red C12 (BODIPY 558/568 C12, MCE, Catalog No. HY-138226) in CM for 16 h, then rigorously washed three times with CM and equilibrated for 1 h to remove extracellular dye aggregates.

Techniques: Labeling, Flow Cytometry, Derivative Assay, Fluorescence

Journal: Molecular Neurodegeneration

Article Title: Alzheimer’s disease protective allele of Clusterin modulates neuronal excitability through lipid-droplet-mediated neuron-glia communication

doi: 10.1186/s13024-025-00840-1

Figure Lengend Snippet: Neuronal CLU facilitates neuron-to-astrocyte lipid transfer. A - B Lipid droplet (LD) staining by LipidTox in day 25 iGlut pure cultures (T/T vs. C/C). n = 6–7 coverslips per group (2 clones per line, 3–4 coverslips for each clone with 4–5 images per coverslip) from two independent differentiations of each line. Representative images in (A) from CD07 line. C - D LipidTox staining of Day 25 iGlut pure cultures after AAV-CLU overexpression. n = 5–6 coverslips per group (one clone from CD05 with genotype C/C, and 4–5 images of each coverslip) from two independent differentiations. Representative images in (C) from CD07 line. E The schematic diagram for the Red C12 lipid transfer assay. F - G Red C12 and LDs (BD493/503) signals in mAst. n = 7–8 coverslips per group (mAst co-cultured with 2 clones per line, 3–5 coverslips for each clone with 4–6 images from each coverslip) from two independent differentiations of each line. Representative images in (F) from CD05 line. H - I A Red C12 transfer assay was performed in the CLU overexpression system. iGlut were first infected with either AAV-eGFP or AAV-hCLU, as in (C). Red C12 was applied to the iGlut on Day 30, and its transfer to mAst was analyzed using the same procedures outlined in (E). n = 6 coverslips per group (mAst co-cultured with one clone of CD05 C/C line, and 6 coverslips for per group with 4–5 images from each coverslip) from two independent differentiations. J The schematic diagram for overexpression of CLU tagged with Flag. K Co-localization of neuron secreted and AAV-derived CLU (Flag +) and LDs (BD493/503 +) in mAst from day- 30 iGlut-mAst cocultures after AAV-hCLU infection (same experimental approach as Fig. G). Representative images from CD07 line. All statistical graphs depict mean ± SEM. Scale bars are indicated in corresponding image. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001

Article Snippet: Day- 30 iGlut were prelabeled by 2.5 μM Red C12 (BODIPY 558/568, Thermo Fisher Scientific, D3835) by adding Red C12 directly into culture medium and incubated for 18 h. iGlut were then washed twice with pre-warmed DPBS and rested for one hour in culture medium in a 37 °C incubator. mAst cells on coverslips and iGlut neurons in culture wells were washed twice with pre-warmed DPBS.

Techniques: Staining, Clone Assay, Over Expression, Cell Culture, Infection, Derivative Assay

Journal: medRxiv

Article Title: Alzheimer’s disease protective allele of Clusterin modulates neuronal excitability through lipid-droplet-mediated neuron-glia communication

doi: 10.1101/2024.08.14.24312009

Figure Lengend Snippet: (A-B) Lipid droplet (LD) staining by LipidTox in day 25 iGlut pure cultures (T/T vs. C/C). n=12 coverslips per group from three independent experiments (2 clones per line, 6 coverslips for each clone with 3-5 cells averaged from each coverslip). (C-D) LipidTox staining of Day 25 iGlut pure cultures after AAV-CLU overexpression. n=6 coverslips per group (one clone of unedited CD07 with genotype T/C, 3-4 cells averaged from each coverslip). (E) The schematic diagram for the Red C12 lipid transfer assay. (F-G) Red C12 and LDs (BD493/503) signals in mAst. n=5-6 coverslips per group (mAst co-cultured with 2 clones per line, 2-3 coverslips for each clone with 3-5 FOV averaged from each coverslip). (H) The schematic diagram for overexpression of CLU tagged with Flag. (I) Co-localization of neuron secreted and AAV-derived CLU (Flag+) and LDs (BD493/503+) in mAst from day-30 iGlut-mAst cocultures after AAV-hCLU infection (same experimental approach as ). Data, mean±SEM. * p <0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: Day-30 iGlut were prelabeled by 2.5 μM Red C12 (BODIPY 558/568, D3835, Thermo Fisher Scientific) by adding Red C12 directly into culture medium and incubated for 18 hours. iGlut were then washed twice with pre-warmed DPBS and rested for one hour in culture medium in a 37 °C incubator. mAst cells on coverslips and iGlut neurons in culture wells were washed twice with pre-warmed DPBS.

Techniques: Staining, Clone Assay, Over Expression, Cell Culture, Derivative Assay, Infection

Journal: The Journal of Reproduction and Development

Article Title: Lipid droplet formation is spatiotemporally regulated in oocytes during follicular development in mice

doi: 10.1262/jrd.2023-055

Figure Lengend Snippet: Emergence of LDs in oocytes during follicular development. (A) Follicles collected from the ovaries of 11-day-old mice were cultured in vitro for 0, 6, and 12 days. Oocytes were isolated from their follicles, stained with BODIPY 493/503, and observed by confocal fluorescence microscopy. (B) Quantification of size (left) and LD-occupied area (right) in oocytes during follicular development. Error bars, mean ± SEM; P-values were determined by an unpaired two-tailed Student’s t -test. * P < 0.05, **** P < 0.0001. n: number of oocytes analyzed. (C) Oocytes collected from the ovaries of 11- or 18-day-old mice were stained with BODIPY 493/503 and observed by confocal fluorescence microscopy. Insets show enlarged images of the representative boxed area. DIC, differential interference contrast. Arrows, LDs. Scale bars: 5 µm.

Article Snippet: For fatty acid (FA) tracking, the follicles were cultured with 10 μM BODIPY 558/568 C12 (Red C12, D3835; Thermo Fisher Scientific) for 2 and 6 days.

Techniques: Cell Culture, In Vitro, Isolation, Staining, Fluorescence, Microscopy, Two Tailed Test

Journal: The Journal of Reproduction and Development

Article Title: Lipid droplet formation is spatiotemporally regulated in oocytes during follicular development in mice

doi: 10.1262/jrd.2023-055

Figure Lengend Snippet: Formation of LDs in the ER during follicular development. (A) Follicles collected from the ovaries of 11-day-old mice were cultured in vitro for 2 or 6 days. Oocytes were isolated from the cultured follicles, stained with BODIPY 493/503 and ER-Tracker Red, and observed by confocal fluorescence microscopy. (B) Follicles collected as described in (A) were cultured in vitro for 2 or 6 days after microinjection of mRNA encoding ACSL3-mCherry. Oocytes were isolated from the cultured follicles, stained with BODIPY 493/503, and observed by confocal fluorescence microscopy. (C) Follicles collected as described in (A) were cultured in vitro in the presence of triacsin C or DMSO as a control for 6 or 12 days. Oocytes were isolated from the cultured follicles, stained with BODIPY 493/503, and observed by confocal fluorescence microscopy. (D) Quantification of size (left) and LD-occupied area (right) in oocytes cultured for 6 or 12 days as described in (C). Error bars, mean ± SEM. P-values were determined by an unpaired two-tailed Student’s t -test. n.s., non-significant, ** P < 0.001, **** P < 0.0001. n: number of oocytes analyzed. (E) Representative electron microscopy images of follicles collected as described in (A) and cultured in vitro for 1 or 6 days. Insets show enlarged images of the boxed area. LD, lipid droplet; ER, endoplasmic reticulum; Mt, mitochondria; Oo, oocyte; Zp, Zona pellucida; GC, granulosa cell; DIC, differential interference contrast. Arrows, LDs. Scale bars: 5 µm.

Article Snippet: For fatty acid (FA) tracking, the follicles were cultured with 10 μM BODIPY 558/568 C12 (Red C12, D3835; Thermo Fisher Scientific) for 2 and 6 days.

Techniques: Cell Culture, In Vitro, Isolation, Staining, Fluorescence, Microscopy, Microinjection, Control, Two Tailed Test, Electron Microscopy

Journal: The Journal of Reproduction and Development

Article Title: Lipid droplet formation is spatiotemporally regulated in oocytes during follicular development in mice

doi: 10.1262/jrd.2023-055

Figure Lengend Snippet: Incorporation of exogenous FAs into intrafollicular oocytes. (A) Follicles collected from the ovaries of 11-day-old mice were co-cultured in vitro with Red C12 for 2 or 6 days. Oocytes were isolated from the cultured follicles, stained with ER-Tracker Green, and observed by confocal fluorescence microscopy. (B) Follicles were collected and cultured as described in (A). Oocytes were isolated from the cultured follicles, stained with BODIPY 493/503, and observed by confocal fluorescence microscopy. (C) Follicles collected from the ovaries of 11-day-old mice were co-cultured in vitro with Red C12 in the presence of triacsin C or DMSO as a control for 6 days. Oocytes were isolated from the cultured follicles and observed by confocal fluorescence microscopy. (D) Follicles collected from the ovaries of 11-day-old mice were co-cultured in vitro with alkyne-OA for 20 h (pulse). Oocytes were isolated from the cultured follicles, subjected to the click reaction, stained with ER-Tracker Red, and observed by confocal fluorescence microscopy (upper panels). Follicles pulsed with alkyne-OA for 20 h were further cultured without alkyne-OA for 6 days (chase), subjected to the click reaction, stained with Lipi-Deep Red, and observed by confocal fluorescence microscopy (bottom panels). Insets show enlarged images of the boxed area. DIC, differential interference contrast. Arrows, LDs. Scale bars: 5 µm.

Article Snippet: For fatty acid (FA) tracking, the follicles were cultured with 10 μM BODIPY 558/568 C12 (Red C12, D3835; Thermo Fisher Scientific) for 2 and 6 days.

Techniques: Cell Culture, In Vitro, Isolation, Staining, Fluorescence, Microscopy, Control

Journal: The Journal of Reproduction and Development

Article Title: Lipid droplet formation is spatiotemporally regulated in oocytes during follicular development in mice

doi: 10.1262/jrd.2023-055

Figure Lengend Snippet: Association between GCs and oocytes via TZPs is involved in LD formation during follicular growth. (A) Schematic representation of the strategy to create follicles with GCs partially or completely removed from the follicles (top). Each follicle was further cultured in vitro for 2 days, stained with BODIPY 493/503 after the removal of GCs, and observed immediately by confocal fluorescence microscopy (bottom). (B) Follicles collected from the ovaries of 11-day-old mice were cultured in vitro in the presence of CBX or PBS as a control for 6 days. Oocytes were isolated from the cultured follicles, stained with BODIPY 493/503 and SiR-Actin to visualize TZPs, and observed by confocal fluorescence microscopy. (C) Quantification of size (left) and LD-occupied area (right) in oocytes cultured as described in (B). Error bars, mean ± SEM; P-values were determined by an unpaired two-tailed Student’s t -test. n.s., non-significant, ** P < 0.001. n: number of oocytes analyzed. (D) Follicles collected from the ovaries of 11-day-old mice were co-cultured in vitro with Red C12 in the presence of CBX or PBS as a control for 6 days. Oocytes were isolated from the cultured follicles and observed by confocal fluorescence microscopy. Insets show enlarged images of the indicated boxed areas. DIC, differential interference contrast. Arrows, LDs. Scale bars: 5 µm.

Article Snippet: For fatty acid (FA) tracking, the follicles were cultured with 10 μM BODIPY 558/568 C12 (Red C12, D3835; Thermo Fisher Scientific) for 2 and 6 days.

Techniques: Cell Culture, In Vitro, Staining, Fluorescence, Microscopy, Control, Isolation, Two Tailed Test