PPs at 4 hours, 24 hours and 7 days were labelled with 0.5 μM BODIPY FL (Thermo Fisher, Waltham, MA), a hydrophobic dye that stains lipids, in PBS for 15 minutes at 4°C. Samples were acquired on a BD Symphony A5 flow cytometer (BD Biosciences, Franklin Lakes, NJ) with forward and side scatter in log mode and thresholding set on BODIPY-positive events. MegaMix-Plus SSC microparticles (Diagnostica Stago, Parsippany, NJ) were used to optimize scatter settings and establish size regions. Data was analysed in FlowJo 10.8 (BD Biosciences, San Jose, CA).
Bodipy fl
Manufactured by Thermo Fisher Scientific
Sourced in United States
BODIPY FL is a fluorescent dye that can be used to label biomolecules. It has an excitation wavelength of 488 nm and an emission wavelength of 515 nm.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (4 mentions)
Zen blue software, by Zeiss (2 mentions)
Alexa fluor 568, by Thermo Fisher Scientific (2 mentions)
Atto488, by ATTO-TEC (2 mentions)
Lsm 700 confocal microscope, by Zeiss (2 mentions)
Lsm 880, by Zeiss (2 mentions)
Monobromobimane mbbr, by Thermo Fisher Scientific (2 mentions)
Symphony a5 flow cytometer, by BD (1 mentions)
Megamix plus ssc microparticles, by Diagnostica Stago (1 mentions)
Cell observer microscope, by Zeiss (1 mentions)
Sypro ruby, by Thermo Fisher Scientific (1 mentions)
A1 confocal microscope, by Nikon (1 mentions)
Ulbp1 pe, by R&D Systems (1 mentions)
Qev size exclusion column, by Izon Science (1 mentions)
Cytofix, by BD (1 mentions)
Fc receptor blocking reagent, by Miltenyi Biotec (1 mentions)
8 hydroxypyrene 1 3 6 trisulfonic acid, by Thermo Fisher Scientific (1 mentions)
Dna aptamer, by Integrated DNA Technologies (1 mentions)
Ni nta agarose superflow resin, by Qiagen (1 mentions)
Atto680, by ATTO-TEC (1 mentions)
32 protocols using bodipy fl
1
Lipid Staining for Flow Cytometry
2
Quantitative Analysis of LDL Uptake
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LDL uptake was measured using using fluorescently labelled Low Density Lipoprotein from Human Plasma, BODIPY® FL complex (Thermo Fisher Scientific # L3483) according to manufacturer’s protocol. In brief, HUVECs were reverse transfected with indicated siRNA oligos and seeded at 1 × 104 on gelatin coated chamber slides. Cells were changed to lipid-deprived media containing 2% lipid depleted serum 24 h before harvest. Cells were labelled with 15 µg/mL BODIPY® FL for 30 min on ice and chased for 10 min at 37 °C. Cells were fixed in 4% paraformaldehyde on ice, washed three times in PBS and counterstained with Hoechst in PBS. The intensity was quantified from an average of 500 cells per experiment and each experiment represent cells pooled from four individual donors. For quantitative analysis, cells were imaged on a Zeiss Cell Observer microscope. The Zen Blue software (Zeiss) was used for automated capture of 40 images per sample with a 20x magnification. A pipeline was created in Cellprofiler39 (link),40 (link) to count dots and to measure intensity per cell. KNIME was used for data mining and analysis41 .
3
Multimodal Histological Staining
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The paraffin embedded histological sections were deparaffinized in absolute xylenes and immersed in 100%, 95% and 70% (v/v) ethanol in a stepwise manner for rehydration. Subsequently, the sections were incubated with select fluorophores in 1× PBS at 37 °C for 30 minutes: BODIPY FL (lipids, Thermo Fisher), SYPRO Ruby (protein, Thermo Fisher), and 4',6-diamidino-2-phenylindole (DAPI) (nuclear material, Thermo Fisher).
4
Visualizing Cellular Fatty Acid Uptake
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HCT116, NTC, and FASN shRNA cells were plated at 10,000 cells/well on an 8-well coverslip u-slide (Ibidi #80826) and treated with CD36 neutralizing antibody (Cayman Chemical #1009893) for 24 h. After incubation with neutralizing antibody, cells were then treated with fluorescent FA analog BODIPY FL (Thermo Fisher #D3822) for 10 min in serum free McCoy's 5A medium supplemented with 10% fatty acid free BSA. Cells were washed twice with PBS and fixed with PBS containing 5% formalin for 20 min at 37°C. Cell were then imaged via confocal microscopy using a Nikon A1 Confocal Microscope.
5
Exosome Isolation and Identification
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Exosomes were isolated from serum using Izon Science qEV Size Exclusion Columns according to the manufacturer's instructions. For identification of exosomes in serum, Fc receptor blocking reagent (Miltenyi) was added to paired serum, exosome and protein fractions before staining with BODIPY-FL (ThermoFisher) and ULBP1-PE (R&D). Samples were fixed with Cytofix (BD) before acquisition. Samples were acquired by ImagestreamXL (Amnis) and data analysis performed using IDEAS software (Amnis).
6
Recombinant Protein Expression and Purification
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Plasmids for recombinant expression of His-GFP and His-SNAP were transformed into Escherichia coli Rosetta 2 cells (Novagen) for protein expression. Cultures were grown in LB at 37 °C to an OD600 of 0.4, then temperature shifted to 16 °C for 20 min and induced using 0.5 mM IPTG at an OD600 between 0.5 and 0.7. Cultures were grown overnight at 16 °C. Cells were harvested by centrifugation and resuspended in PBS. Cells were lysed with 3 cycles of sonication and freeze–thaw and clarified by centrifugation. The supernatants were incubated with Ni-NTA agarose superflow resin (Qiagen) for 1 h at 4 °C. After extensive washing, proteins were eluted in a buffer containing 150 mM NaCl, 25 mM Tris, 250 mM imidazole, pH 8, 10% glycerol, and then dialyzed overnight to remove imidazole.
DNA aptamer (GAA GAC GAG CGG CGA GTG TTA TTA CGC TTG GAA ACA ACC CC) was purchased from Integrated DNA Technologies and contained a 5′ BG and 3′ FAM fluorophore modifications. Fluorescent dyes, BODIPY-FL and 8-Hydroxypyrene-1,3,6-Trisulfonic Acid, Trisodium Salt or pyranine (HPTS) were purchased from ThermoFisher Scientific. HPLC purified doxorubicin hydrochloride was purchased from Sigma-Aldrich. BG-GLA-NHS for the functionalization of the 3′-amino aptamer was purchased from New England Biolabs.
DNA aptamer (GAA GAC GAG CGG CGA GTG TTA TTA CGC TTG GAA ACA ACC CC) was purchased from Integrated DNA Technologies and contained a 5′ BG and 3′ FAM fluorophore modifications. Fluorescent dyes, BODIPY-FL and 8-Hydroxypyrene-1,3,6-Trisulfonic Acid, Trisodium Salt or pyranine (HPTS) were purchased from ThermoFisher Scientific. HPLC purified doxorubicin hydrochloride was purchased from Sigma-Aldrich. BG-GLA-NHS for the functionalization of the 3′-amino aptamer was purchased from New England Biolabs.
7
Fluorescent Dye Preparation Protocol
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The eight fluorophores utilized in this study were obtained commercially in their succinimidyl ester form including Fluorescein, BODIPY FL, AlexaFluor568, AlexaFluor647 (Thermo Fisher Scientific), ATTO488, ATTO680 (ATTO-TEC), Cy3B and Cy5 (GE Healthcare Life Sciences).
8
Quantifying Macrophage Efferocytosis of Neutrophils
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Biogel-elicited macrophages were collected from WT mice, as previously described [19 (link)], and were seeded into 24-well plates at 0.5 × 106 cells/well and allowed to adhere. Exudated neutrophils from WT and Gal-3–null mice were collected after 4 h zymosan-induced peritonitis and labeled with BODIPY-FL (Thermo Fisher Scientific) for 5 min at 37°C. After washing, neutrophils were resuspended in RPMI 1640 at 2 × 106/ml, and 0.5 ml was added to each well of macrophages and incubated for 1 h. After extensive washing efferocytosis was quantified using ImageJ software (U.S. National Institutes of Health, Bethesda, MD, USA). Images were split into their individual red/blue/green channels, and the same background threshold was applied to all images before quantification of fluorescence.
9
Lipid Droplet Quantification in Morula Embryos
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For lipid droplets quantification, morula-stage embryos were preferred and selected over blastocysts because the absence of fluid-filled cavity allows a better imaging resolution. A first staining of morula embryos was performed using 300 nM MitoTracker Orange CMTMRox (ThermoFisher Scientific, Burlington, ON, Canada) in a flushing medium (ViGRO, Bioniche, Belleville, ON, Canada) at 37°C during 30 min. Then, a second staining was performed using 3 μg/mL Bodipy FL (ThermoFisher Scientific) and 1 μg/mL Hoechst blue dye 33342 (ThermoFisher Scientific) diluted in flushing medium and incubated during 10 min at 37°C. Finally, embryos were fixed in 4% paraformaldehyde (Sigma-Aldrich, Oakville, ON, Canada) for 15 min at room temperature, washed 3 times in flushing medium, and mounted on a microscope slide using secure Seal imaging spacer (Sigma-Aldrich).
10
Cellular Transfection Efficiency Analysis
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2 × 106 RAW264.7, HL-60, EL4 cells, primary macrophages or splenocytes were seeded in 6-well plates in DMEM medium containing 10% FBS. The next day, cells were washed with PBS and treated or untreated with 1 ml LNP (labeled with a green fluorescent probe Bodipy-FL, ThermoFisher scientific, D-3792) at different concentrations containing or not, a fluorescent siRNA (labeled with fluorescent probe cyanine-3, Sigma, SIC003) at 37°C in DMEM medium with 10% FBS. The percentage of positive Bodipy-FL and cyanin-3 cells, and the mean of fluorescence intensity (MFI) were analyzed by flow cytometry on BD LSR FortessaTM (BD Biosciences) 24 h post-transfection using the fluorescein isothiocyanate (FITC530/30-A) and the cyanine-3 575/26-A) channels.
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