Thawed PBMCs were incubated for 30 min with 0.2 µg/ml BODIPY dye (ThermoFisher Scientific) and subsequently stained and analyzed by flow cytometry, as described above. The difference of mean fluorescence intensity (MFI) between BODIPY stained and all-but-BODIPY-stained samples (ΔMFI) served as a measure of lipid droplet content.
Bodipy dye
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
BODIPY dye is a fluorescent label used in various biological applications. It is a synthetic organic compound that can be used to label and track biomolecules, such as proteins, lipids, and nucleic acids, in living cells or in vitro systems. The BODIPY dye has a high quantum yield, making it a useful tool for fluorescence-based experiments and imaging techniques.
Automatically generated - may contain errors
Lab products found in correlation
Cytation 5 plate reader, by Agilent Technologies (2 mentions)
Bodipy 581 591 c11 reagent, by Thermo Fisher Scientific (2 mentions)
Bodipy, by Thermo Fisher Scientific (1 mentions)
Triglyceride solution, by Thermo Fisher Scientific (1 mentions)
Epifluorescence microscopy, by Olympus (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Primary rabbit polyclonal antibodies, by Abcam (1 mentions)
Alexa fluor 594 goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
Vectashield medium, by Vector Laboratories (1 mentions)
Hematoxylin and eosin, by Wuhan Servicebio Technology (1 mentions)
11 protocols using bodipy dye
1
Quantifying Lipid Droplet Content
2
Bodipy Staining of Differentiated Cells
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BA were differentiated with medium 1 or 2 and incubated with 1–5 μg/mL of Bodipy dye (D3835, Thermo Fisher Scientific) dilution for 30 min at 37 °C. The cells were washed twice with PBS, fixed with 4% PFA at pH 7.5 for 20 min at room temperature, and then incubated with DAPI (1 μg/mL) for 5 min at room temperature. Coverslips were adhered with antifade mounting medium, allowed to dry overnight, and then maintained at 4 °C until confocal microscopy analysis.
3
Muscle-Derived Progenitor Cell Assays
4
BODIPY Dye Labeling Protocol
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The green BODIPY dye was purchased from Thermo Fischer Scientific (Massachusetts, USA) CAS: 121207-31-6. Stock solution concentration was 1 mg·mL−3; the solvent was anhydrous DSMO, from which 10 µL was added to 1 mL of cell suspension. The incubation time was 30 min; the tube was placed in a dark place, at lab temperature. After that, the cells were rinsed with 1 mL PBS buffer two times (centrifugation 8000× g for 5 min, room temperature), and then 1 mL of PBS buffer was added in which the pellet was resuspended. This procedure was followed when preparing samples for both flow cytometry and fluorescent microscopy.
When executing flow cytometry experiments, the samples were illuminated by a 488 nm laser; fluorescence was then captured in the same channel as autofluorescence, i.e., 535 ± 35 nm.
When executing flow cytometry experiments, the samples were illuminated by a 488 nm laser; fluorescence was then captured in the same channel as autofluorescence, i.e., 535 ± 35 nm.
5
Lipid Peroxidation Measurement in ASCs
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Lipid peroxidation refers to the degradation of cellular lipids due to the generation of reactive oxygen species within the cell. For the determination of lipid peroxidation, a sensitive fluorescent reporter BODIPY 581/591 C11 reagent (BODIPY dye, Invitrogen) was used. The phenylbutadiene portion of the dye allows the shift of fluorescence emission peak from 590 nm-red to 510 nm-green upon oxidation. To determine the lipid peroxidation, after different treatments, ASCs cultured in a 6-well plate were incubated with 10 μM BODIPY reagent for 30 min at 37 °C. The ratio of reduction (590 nm)/oxidation (510 nm) was derived by reading the fluorescence intensities at the separate wavelengths on Cytation 5 plate reader (Biotek) while the percent lipid peroxidation was analyzed by the flow cytometer.
6
Glutamate-Induced Lipid Peroxidation Assay
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Lipid peroxidation after 8 h of glutamate exposure was analyzed by staining with 2 μM BODIPY dye (Invitrogen) for 60 min at 37 °C. A shift in BODIPY fluorescence from red to green was assessed by excitation at 488 nm and detection with a 525/30 nm band pass filter and a 690/50 nm band pass filter. Data were recorded from 1×104 cells in triplicate per condition.
7
Quantifying Drosophila Total Body Lipids
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The whole fly bodies were collected and fixed in 4% paraformaldehyde for 30 min at room temperature. After washing with phosphate-buffered saline, the flies were repeatedly frozen in liquid nitrogen and thawed on ice three times, followed by staining with a solution containing 1 μg/ml BODIPY dye (Invitrogen, Darmstadt, Germany) for 1 h in the darkness before observation by epifluorescence microscopy (Olympus, Hamburg, Germany).
Total body triacylglycerols (TAGs) in flies were determined using a coupled colorimetric assay method as described previously (Hildebrandt et al., 2011 (link); Hoffmann et al., 2013 (link); Li et al., 2016 (link)). Briefly, eight males (or five females) per group were weighed and homogenized in 1 ml 0.05% Tween-20 using a Bead Ruptor 24 (BioLab Products, Bebensee, Germany). Homogenates were heat-inactivated for 5 min at 70°C and incubated with triglyceride solution (Fisher Scientific, Waltham, MA, USA) at 37°C for 30 min with mild shaking. The absorbance was read at 562 nm and glyceryl trioleate served as a TAG standard.
Total body triacylglycerols (TAGs) in flies were determined using a coupled colorimetric assay method as described previously (Hildebrandt et al., 2011 (link); Hoffmann et al., 2013 (link); Li et al., 2016 (link)). Briefly, eight males (or five females) per group were weighed and homogenized in 1 ml 0.05% Tween-20 using a Bead Ruptor 24 (BioLab Products, Bebensee, Germany). Homogenates were heat-inactivated for 5 min at 70°C and incubated with triglyceride solution (Fisher Scientific, Waltham, MA, USA) at 37°C for 30 min with mild shaking. The absorbance was read at 562 nm and glyceryl trioleate served as a TAG standard.
8
BODIPY Adipocyte Differentiation Assay
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BODIPY staining was performed to detect adipocyte differentiation, which is a fluorescence-based approach. Adipocytes were fixed with 4% paraformaldehyde for 30 min at room temperature and then incubated with BODIPY dye (Invitrogen) (working solution 1 mg/mL) for 30 min. After incubation, cells were treated with 4’,6-diamidino-2-phenylindole (DAPI) (Gibco, Grand Island, NY, USA) to stain the nuclei for 10 min. Immunofluorescence images were captured by an Olympus IX71 microscope (OLYMPUS, Dalian, China).
9
Immunofluorescence Imaging of SNAP23, BSCL2, and COPA
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For immunofluorescence staining, cells cultured on glass cover slips were fixed in 4 % paraformaldehyde in PBS (w/v) for 10 min at room temperature, followed by washing in PBS. The cells were then treated with 0.1 % Triton X-100 in PBS (v/v) for 5 min, washed three times in PBS and blocked for 30 min in 3 % bovine serum albumin (w/v). Primary rabbit polyclonal antibodies (Abcam) against the SNAP23, BSCL2 or COPA proteins were used. Specificity of antibodies was verified by western blot analysis. The cells were incubated overnight with primary antibodies in 1:100 dilution at 4 °C. After three washes in PBS cells were incubated for 1 h with a secondary antibody — goat anti-rabbit Alexa Fluor 594 (Invitrogen), diluted 1:200. After further washing in PBS cells were stained with BODIPY dye (Invitrogen) to visualise lipid droplets. Finally, nuclei were counterstained with DAPI in the Vectashield medium (Vector Laboratories).
10
Muscle Histological Analysis Protocols
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For HE staining, GA muscles were fixed with paraformaldehyde (4%), dehydrated, and paraffin-embedded. Cross-sectional 5 μm thick slices of muscle were prepared and stained with hematoxylin and eosin (Servicebio) to observe the morphological changes. And cross-sectional areas were analyzed using Image J processing software.
For SDH and BODIPY staining, fresh GA muscles were embedded in OCT after isolation, then snap-frozen by isopentane with liquid nitrogen. 8-μm thick slices were prepared and stained with SDH solution (Solarbio) or BODIPY dye (3.8 μM working solution, Invitrogen).
For SDH and BODIPY staining, fresh GA muscles were embedded in OCT after isolation, then snap-frozen by isopentane with liquid nitrogen. 8-μm thick slices were prepared and stained with SDH solution (Solarbio) or BODIPY dye (3.8 μM working solution, Invitrogen).
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