Cells were seeded onto dishes and fixed with 4% paraformaldehyde (Beyotime, Shanghai, China) for 20 min. After incubation in the fluorescent lipophilic dye BODIPY 493/503 solution (5 ng/mL, Thermo Fisher) for 1 h at 37°C in the dark, images were generated using an Olympus FV‐1000 confocal microscope (Olympus Corporation, Tokyo, Japan). Results are expressed as relative fluorescence, which was quantified using the ImageJ software.
Bodipy 493 503 solution
Manufactured by Thermo Fisher Scientific
Sourced in Japan
BODIPY 493/503 solution is a fluorescent dye commonly used in biological research applications. It exhibits green fluorescence and is commonly used to stain lipid droplets and other lipid-rich structures within cells.
Automatically generated - may contain errors
Lab products found in correlation
Paraformaldehyde (pfa), by Beyotime (1 mentions)
Fv1000 confocal microscope, by Olympus (1 mentions)
Dmi4000b inverted microscope, by Leica (1 mentions)
Nile red, by Merck Group (1 mentions)
Application suite las software, by Leica (1 mentions)
Calcofluor white m2r, by Merck Group (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Cm 0602, by Procell (1 mentions)
Pyg0040, by Boster Bio (1 mentions)
Oxidized ldl, by Thermo Fisher Scientific (1 mentions)
4 protocols using bodipy 493 503 solution
1
Lipid Quantification in Cells
2
Lipid and Cell Wall Staining Protocol
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After 72 h of shake flask culture, 100 μL of cell culture was transferred to a 1.5-mL Eppendorf tube, centrifuged, and washed with 1 mL of deionized water. Cells were then centrifuged at 3,000 × g for 5 min and resuspended in 100 μL of PBS. Resuspended cells were treated with 1 μL of BODIPY 493/503 solution (1 mg·mL−1 in ethanol; Thermo Fisher Scientific), 3 μL Nile Red (1 mg·mL−1 in DMSO), or 0.5 μL Calcofluor White M2R (1 mg·mL−1 stock solution; Sigma-Aldrich) and kept at 4 °C in the dark for 10 min. Fluorescent microscope pictures were analyzed using a Leica DMI4000B inverted microscope and processed with Leica Application Suite (LAS) software.
3
Lipid Regulation in AML-12 Mouse Hepatocytes
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Alpha mouse liver 12 (AML-12) cell line was derived from mouse hepatocytes (CD1 strain, line MT42) that carry a human TGF-α transgene. AML-12 cells were maintained in DMEM/F12 containing 10% fetal bovine serum, 10 μg/ml insulin, 5.5 μg/ml transferrin, 5 ng/ml selenium, 40 ng/ml dexamethasone, and 1% penicillin/streptomycin (Procell #CM-0602) and cultured at 37°C with 5% CO2. Cells were seeded into 12-well plates at a density of 7.5E+4 cells per well. After 12 h, cells were transfected by a mix of 50 nM Plin3 siRNA (Tsingke Biotechnology; targeting sequence: GGCTCAAGAAATGGTATCT) or control siRNA (sense: UUCUCCGAACGUGUCACGUTT; anti-sense: ACGUGACACGUUCGGAGAATT) and Lipofectamine 3000 (Invitrogen #L3000008). After 36 h of RNA interference, cells were treated with 200 μM oleic acid (Sigma #O1008) and DMSO vehicle (Boster #PYG0040) for 12 h. Cells were extracted for RNA (n = 3 wells, repeated twice), protein (n = 3 wells, repeated once), and Bodipy staining (n = 3 wells, repeated once). Results were reproducible. For Bodipy staining, cells were fixed with 4% paraformaldehyde for 15 min, stained with 10 nM Bodipy 493/503 solution (Invitrogen #D3922) for 10 min, and then stained with DAPI (Beyotime #C1005) for 5 min. Cells were imaged with a fluorescence microscope (Nikon Ni-U).
4
Lipid Phagocytosis Assay of Immune Cells
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Sorted cells (around 8 × 103 cells for CD45low Lilrb4+ and CD45high Lilrb4+ subsets and 2 × 104 for CD45+ Lilrb4− subset) were collected after centrifugation and distributed in two wells, for control cells and cells fed with Ox-LDL, of a chambered coverslip with 8 wells (Ibidi Cat No. 80826) for cell culture and immunofluorescence, in 250 ml per well of RPMI 1640 supplemented with 10% FCS 1% L-Glutamine and 1% Penicillin/Streptomycin. For lipid phagocytosis assay, cells were incubated for 18 h in the presence of 25 mg/ml oxidized-LDL (Invitrogen Cat No. L34357) and then washed once with PBS by centrifugation, in order not to lose the non-adherent cells. For lipid droplets visualization due to ox-LDL ingestion, cells were incubated with 1 mg/ml of Bodipy 493/503 solution (Invitrogen Cat No. D3922) for 30 min at 37 °C, to then visualize under the microscope by using mounting media containing DAPI. Images were taken at 40x magnification.
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