To assess LD size and purity, the different-sized LD fractions isolated from livers of rats fed control, ethanol, or betaine-supplemented ethanol diet were stained with 1,3,5,7-Tetramethyl-8-phenyl-4,4-difluoroboradiazaindacene (BODIPY 493/503; Invitrogen, Carlsbad, CA, USA), as previously described [21 (link),22 (link)]. Briefly, 20 µL of each isolated LD fraction was placed on a slide and stained with 1 µg BODIPY 493/503 (Invitrogen, Carlsbad, CA, USA). All LD fractions were used undiluted, except for LD1 and LD2 fractions obtained from livers of ethanol-fed rats, which were diluted 1:20 and 1:4, respectively. After staining for 5 min, LDs were visualized under a Keyence BZ-X810 florescence microscope and images were captured. LD size was quantified using Keyence BZ-X810 Analyzer software [22 (link)].
Bodipy 493 503
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany, Italy, China, United Kingdom, Japan, France, Switzerland, Belgium
BODIPY 493/503 is a fluorescent dye that can be used to stain neutral lipids and lipid droplets. It has an excitation maximum at 493 nm and an emission maximum at 503 nm.
Automatically generated - may contain errors
Lab products found in correlation
Hoechst 33342, by Thermo Fisher Scientific (79 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (64 mentions)
Oil red o, by Merck Group (25 mentions)
Lsm 780 confocal microscope, by Zeiss (22 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (21 mentions)
Lsm 700, by Zeiss (16 mentions)
Lsm 800, by Zeiss (14 mentions)
Lsm 700 confocal microscope, by Zeiss (14 mentions)
Triton x 100, by Merck Group (13 mentions)
Nile red, by Merck Group (12 mentions)
Hoechst 33342, by Merck Group (12 mentions)
Bodipy 558 568 c12, by Thermo Fisher Scientific (11 mentions)
Mitotracker red cmxros, by Thermo Fisher Scientific (10 mentions)
Lsm800 confocal microscope, by Zeiss (10 mentions)
Dexamethasone (dex), by Merck Group (10 mentions)
Vectashield, by Vector Laboratories (10 mentions)
Fluorescence microscope, by Olympus (9 mentions)
Lsm 780, by Zeiss (9 mentions)
Oleic acid, by Merck Group (9 mentions)
Mitotracker deep red, by Thermo Fisher Scientific (9 mentions)
909 protocols using bodipy 493 503
1
Lipid Droplet Characterization in Ethanol-Fed Rats
2
Adipocyte Lipid Visualization and Larval Imaging
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The 3T3-L1 preadipocytes were grown on coverslips and followed by treatment according to the experimental plan. After being treated for the appropriate times, the cells were immobilized with 4% paraformaldehyde (PFA) for 15 min and then permeabilized with 0.1% Triton X-100 (Sigma-Aldrich, USA) for 10 min at room temperature. Then, cells were stained with BODIPY 493/503 (used at 1:1000 dilution, Invitrogen, USA) for 10 min to and with DAPI (used at 1:1000 dilution, Invitrogen, USA) for 5 min. Confocal microscopy was used with ZEN software to acquire high-quality images. The larvae were maintained in RO water containing BODIPY 493/503 (used at 1:500 dilution, Invitrogen, USA) for 30 min and then anesthetized with MS-222 (Sigma-Aldrich, USA). Images were acquired by fluorescence stereo microscope (Olympus RI2, Japan).
3
Staining Lipid Droplets with BODIPY in Yeast, HeLa Cells, and Worms
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When staining LDs with BODIPY, yeasts cells in early stationary growth phase were washed with phosphate buffered saline and incubated with 0.5 μg/ml BODIPY 493/503 (Invitrogen) for 10 min.
To induce LDs, Hela cells were incubated with 300 μM BSA-oleic acid (Sigma # O3008) for 14–18 h, stained with 5 μg/ml BODIPY for two h and washed with PBS. Cells were shifted to CO2-independent medium (Gibco) containing 10% FBS and 2 mM L-glutamine before live-cell imaging.
Worms were age matched to 23 h post-L4 stage, 10–15 worms were incubated in BODIPY493/503 (Invitrogen) at 6.7 ug/mL in M9 Buffer for 20 min, followed by 3 wash cycles in M9 Buffer. They were immediately mounted in M9 Buffer for confocal imaging on a Nikon (Garden City, NY) E800 spinning disk confocal microscope using MetaMorph imaging software.
To induce LDs, Hela cells were incubated with 300 μM BSA-oleic acid (Sigma # O3008) for 14–18 h, stained with 5 μg/ml BODIPY for two h and washed with PBS. Cells were shifted to CO2-independent medium (Gibco) containing 10% FBS and 2 mM L-glutamine before live-cell imaging.
Worms were age matched to 23 h post-L4 stage, 10–15 worms were incubated in BODIPY493/503 (Invitrogen) at 6.7 ug/mL in M9 Buffer for 20 min, followed by 3 wash cycles in M9 Buffer. They were immediately mounted in M9 Buffer for confocal imaging on a Nikon (Garden City, NY) E800 spinning disk confocal microscope using MetaMorph imaging software.
4
Lipid Content Visualization in NSCLC Cells
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The lipophilic fluorescence dye BODIPY 493/503 (Invitrogen, Waltham, MA, USA) was used to monitor the content of neutral lipids in NSCLC cells. After being fixed in 4% PFA for 20 min, cells were incubated with BODIPY 493/503 (D3299, Thermo Fisher, Waltham, MA, USA) and DAPI in PBS at RT for 15 min. Finally, the cells were visualized with a fluorescence microscope (Olympus, Tokyo, Japan). A representative image is shown from three independent experiments.
5
Multi-Staining of Cellular Components
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Before staining various cell components, cells were fixed with 4% paraformaldehyde in PBS (for 20 min at 20 °C), permeabilized with 0.5% Triton X-100 in PBS (for 15 min at 20 °C), and a saturation was performed using 2% BSA in PBS (for 30 min at 20 °C). Cells were washed three times with PBS after each step.
To delineate cell shape, actin was stained using 4 U/mL Rhodamine Phalloïdin (Invitrogen) in PBS. Cells were incubated for 30 min at 37 °C with Rhodamine Phalloïdin, then washed five times with PBS.
To observe lipid droplets, they were stained using 3 μg/mL Bodipy 493/503 (Molecular Probes) in PBS. Cells were incubated for 15 min at 20 °C with Bodipy 493/503, then washed five times with PBS.
To detect cell nuclei, 2 μg/mL DAPI (Invitrogen) was added to the Citifluor (Biovalley) antifading solution used for confocal microscopy observations.
Once the various cell markings were performed, the collagen gel carrying the cells was placed between a slide and a coverslip in Citifluor and cells were observed by confocal microscopy under a Zeiss LSM 700 confocal microscope at a 40× magnification (MIMA2 platform, INRA).
To delineate cell shape, actin was stained using 4 U/mL Rhodamine Phalloïdin (Invitrogen) in PBS. Cells were incubated for 30 min at 37 °C with Rhodamine Phalloïdin, then washed five times with PBS.
To observe lipid droplets, they were stained using 3 μg/mL Bodipy 493/503 (Molecular Probes) in PBS. Cells were incubated for 15 min at 20 °C with Bodipy 493/503, then washed five times with PBS.
To detect cell nuclei, 2 μg/mL DAPI (Invitrogen) was added to the Citifluor (Biovalley) antifading solution used for confocal microscopy observations.
Once the various cell markings were performed, the collagen gel carrying the cells was placed between a slide and a coverslip in Citifluor and cells were observed by confocal microscopy under a Zeiss LSM 700 confocal microscope at a 40× magnification (MIMA2 platform, INRA).
6
Quantifying Adipocyte Lipid Content
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3T3-L1 cells were trypsinized, washed with PBS, stained with BODIPY 493/503 (Molecular Probes, 1 µg/ml final) for 5 min, and analyzed via flow cytometry (BD CantoII or Beckman Coulter Epics XL-MCL). GLUT4myc7-GFP-assays were performed as described previously36 (link). Intracellular GLUT4 (3G10A3, Thermo Fisher) staining of 3T3-L1 cells was performed after treatment with FACS Lysing Solution and Permeabilizing Solution 2 (BD) according to manufacturer’s instructions. For epifluorescence microscopy (Zeiss Axiovert 200) using AxioVision software, cells were fixed (4% PFA), permeabilized (0.2% Triton X-100), and stained with BODIPY 493/503 (1 µg/µl, Thermo Fisher), DAPI (0.2 µg/µl, Sigma-Aldrich) and Phalloidin (1:200, Santa Cruz). At least 15,000 cells per sample were analyzed.
7
Visualizing Mitochondria and Lipids in 3T3-L1 Cells
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3T3-L1 cells were grown as described in ‘3T3-L1 culture, differentiation and treatment’. At day 7 post induction cells were incubated in a medium containing 50 μM of Mitotracker (Cell Signaling, 9082P) for 30 min. Cells were then washed with PBS and fixed with 4% formaldehyde during 10 min and rinsed 3 times with PBS. Lipid droplets were stained with a Bodipy 493/503 (D3922, Thermo Fisher Scientific) in a staining solution (1 μg/ml Bodipy 493/503, 150 mM NaCl) for 10 min at room temperature. Nuclei were stained with DAPI (0.5 μg/ml).
8
Lipid Staining in Caenorhabditis elegans
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Embryo lipid staining was performed as described previously [33 (link)]. In short, embryos were obtained by alkaline hypochlorite treatment of adult hermaphrodites, transferred to PCR tubes (Eppendorf, Hamburg, Germany) containing 4% formaldehyde in M9 buffer and subjected to 3 freeze-thaw cycles in liquid nitrogen and a 37°C water bath. Embryos were then incubated with 1 μg/ml BODIPY 493/503 (Thermo Fisher Scientific) in M9 buffer for 1 hour and washed 3 times with M9 + 0.01% Triton X-100 (Sigma #9001-93-1). Next, nuclei were stained with 5 ng/mL DAPI and imaged by epifluorescence microscopy.
Adults were fixed for lipid staining by incubating in 4% paraformaldehyde (Sigma-Aldrich 158127) for 1 hour at room temperature. Adults were then washed twice with PBS, resuspended in 95% ethanol and washed again in PBS. Next, fixed animals were incubated with 1 μg/ml BODIPY 493/503 (Thermo Fisher Scientific) in M9 buffer for 1 hour and washed 3 times with M9 + 0.01% Triton X-100. Animals were stained with 5 ng/mL DAPI and imaged by epifluorescence microscopy.
Adults were fixed for lipid staining by incubating in 4% paraformaldehyde (Sigma-Aldrich 158127) for 1 hour at room temperature. Adults were then washed twice with PBS, resuspended in 95% ethanol and washed again in PBS. Next, fixed animals were incubated with 1 μg/ml BODIPY 493/503 (Thermo Fisher Scientific) in M9 buffer for 1 hour and washed 3 times with M9 + 0.01% Triton X-100. Animals were stained with 5 ng/mL DAPI and imaged by epifluorescence microscopy.
9
Quantifying Lipid Content in BAL Cells
10
Placental Cell Lipid Staining Protocols
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Placental cells were stained for flow cytometry as described above. Cells were incubated in 2 ng/ml BODIPY 493/503 (Thermo Fisher Scientific) in PBS for 20 min at 4°C. Cells were washed in FACS buffer and acquired on a Cytek Aurora (Cytek).
FACS-isolated PAMM1a and PAMM1b were incubated in 250 ng/ml BODIPY 493/503 (Thermo Fisher Scientific) in PBS for 1 h at 37°C, fixed in 4% paraformaldehyde solution (Sigma-Aldrich), and washed twice in PBS. Cytospins were prepared and mounted using VECTASHIELD Antifade Mounting Medium with DAPI (Vector Laboratories) and imaged using a Zeiss SP8 confocal LSM 700.
FACS-isolated PAMM1a and PAMM1b were incubated in 250 ng/ml BODIPY 493/503 (Thermo Fisher Scientific) in PBS for 1 h at 37°C, fixed in 4% paraformaldehyde solution (Sigma-Aldrich), and washed twice in PBS. Cytospins were prepared and mounted using VECTASHIELD Antifade Mounting Medium with DAPI (Vector Laboratories) and imaged using a Zeiss SP8 confocal LSM 700.
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