For confocal microscopy, macrophages were seeded in black poly-d-lysine coated glass 96-well plates (MatTek Corporation, Ashland, MA, USA). To stain lysosomes, macrophages were incubated with 75 nM Lysotracker Red or Deep Red (Thermo Fisher Scientific) at 37°C/5%CO2 for 1 h before fixation. Cells were fixed for 1 h in 1% EM-grade formaldehyde, followed by quenching with PBS/1.5 mg/ml glycine for 10 min and blocking in 5% human serum for 45 min, all at room temperature. For immunostaining, cells were permeabilized for 10 minutes with 0.1% Triton X-100 before blocking and subsequently stained with primary and secondary antibodies for 30 minutes each in the dark at room temperature. Finally, cells were stained with phalloidin-Alexa488 (Thermo Fisher Scientific) and/or LipidTOX Green (Thermo Fisher Scientific) for 30 min according to the manufacturers’ instructions, and/or 50 μg/ml Filipin complex from Streptomyces filipinensis (Sigma-Aldrich) for 2 h at room temperature in the dark. Lysotracker and filipin pictures were taken using a SP8WLL confocal microscope (Leica, Amsterdam, The Netherlands). Galectin-3 and NDP52 colocalization was visualized using a Dragonfly spinning-disk confocal microscope (Andor Technologies, Belfast, UK) equipped with 405, 488, 561 and 640nm lasers and a Zyla 4.2 sCMOS camera.
Lipidtox green
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom, Germany
LipidTOX Green is a fluorescent dye that can be used to detect and quantify neutral lipids in cells. It binds specifically to neutral lipids, providing a direct measurement of lipid content.
Automatically generated - may contain errors
Lab products found in correlation
Deltavision deconvolution microscope, by GE Healthcare (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Lipidtox deep red, by Thermo Fisher Scientific (2 mentions)
Rnaimax, by Thermo Fisher Scientific (2 mentions)
Chondrogenesis differentiation kit, by Thermo Fisher Scientific (2 mentions)
Phalloidin alexa488, by Thermo Fisher Scientific (1 mentions)
Filipin complex from streptomyces filipinensis, by Merck Group (1 mentions)
Sp8 wll confocal microscope, by Leica (1 mentions)
Wortmannin, by Cayman Chemical (1 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (1 mentions)
Fugene hd, by Promega (1 mentions)
Floid cell imaging station, by Thermo Fisher Scientific (1 mentions)
Formaldehyde, by Electron Microscopy Sciences (1 mentions)
Slowfade gold, by Thermo Fisher Scientific (1 mentions)
Deltavision core image restoration microscope, by GE Healthcare (1 mentions)
Hoecsht 3342, by Cell Signaling Technology (1 mentions)
Stempro osteogenesis differentiation kit, by Thermo Fisher Scientific (1 mentions)
Alcian blue, by Merck Group (1 mentions)
Adipogenesis differentiation kit, by Thermo Fisher Scientific (1 mentions)
Alizarin red, by Abcam (1 mentions)
16 protocols using lipidtox green
1
Confocal Microscopy of Macrophage Lysosomes
2
Lipid Quantification in LNCaP Cells
3
Lipid Quantification in LNCaP Cells
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LNCaP cells were plated in 10% CSS onto acid-etched, poly-D-lysine
coated coverslips and allowed to attach for 24 hours before treatments. Cells
were treated for 96 hours, then washed with ice cold PBS and fixed with 4%
paraformaldehyde. Sodium borohydride was used to quench autofluorescence derived
from residual paraformaldehyde. Cells were stained using DAPI (Sigma) for nuclei
and LipidTOX green (ThermoFisher) for neutral lipids. Imaging was performed with
the GE Deltavision deconvolution microscope. Lipid staining was quantified using
ImageJ.
coated coverslips and allowed to attach for 24 hours before treatments. Cells
were treated for 96 hours, then washed with ice cold PBS and fixed with 4%
paraformaldehyde. Sodium borohydride was used to quench autofluorescence derived
from residual paraformaldehyde. Cells were stained using DAPI (Sigma) for nuclei
and LipidTOX green (ThermoFisher) for neutral lipids. Imaging was performed with
the GE Deltavision deconvolution microscope. Lipid staining was quantified using
ImageJ.
4
Live-cell Imaging of Lipid Droplets
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Hep3B and U2OS cells were cultured at a 37 °C with 5% CO2 and in growth media consisting of MEM GlutaMax or Dulbecco’s modified Eagle’s medium (DMEM) GlutaMax supplemented with 10% fetal bovine serum (FBS) and antibiotic-antimycotic (Thermo Fisher Scientific), respectively. Two days prior to live-cell imaging experiments, 50,000 cells were seeded on 3.5 cm imaging dishes and grown to 40% confluency prior to transfection with the indicated plasmids using FuGENE HD (Promega). In some cases, cells were also incubated for 2 days before imaging with a siRNA mixture, consisting of 5 μl of RNAiMax (Thermo Fisher Scientific) and 2.5 pmol of the specified siRNA. Cells were induced to form LDs by supplementing the growth media with 200 μM OA (dissolved in ethanol) for 4 or 24 h (as indicated), before exchanging the media with either normal growth media or EBSS for 1, 4, or 18 h (as indicated) prior to imaging. LDs were identified by incubating with the LD-specific dyes Bodipy-C12-568 (Thermo Fisher Scientific), LipidTox Green (Thermo Fisher Scientific), or LipidTox Deep Red (Thermo Fisher Scientific) at volume ration of 1:10,000 for 30 min prior to imaging. In some cases, cells were treated with 1 μM Wortmannin (Cayman Chemical Company) for 30 min prior to imaging.
5
Lipid Accumulation Assay in Cancer Cells
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HCT116, SW620, and HCT8 cells were plated at a concentration of 30,000 cells/well in a 48-well plate, treated for 16 h with 5 and 10 μmol/L spiperone—or 5 μmol/L fluoxetine, as a positive control—and stained with 1× LipidTOX Green (Thermo Fisher Scientific). Then, nuclei were stained using Hoechst 33342 (5 μg/mL) and plates were incubated for 30 min in the dark at 37 °C. Subsequently, cells were washed with PBS and fixed with 4% paraformaldehyde for 15 min in the dark. Signals were acquired with a fluorescence microscope (FLoid Cell Imaging Station, Life Technology, Carlsbad, CA, USA), and images were analyzed using ImageJ software v 1.52a.
6
Quantifying Adipocyte Lipid Droplets via Microscopy
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Following differentiation, media was aspirated and 4% formaldehyde (Electron Microscopy Sciences) in PBS was immediately added for 30 min at room temperature. Excess paraformaldehyde was quenched with 100 mM ammonium chloride. Non-specific antibody binding was blocked by pre-incubating for 30 min in 2% bovine serum albumin in PBS/0.01% saponin (which was also used as an antibody diluent) at room temperature. Anti-perilipin antibody (GP-29, Progen) was diluted at a 1:1000 concentration in antibody diluent and incubated overnight at 4 °C. Subsequently, coverslips were washed with PBS and incubated with secondary antibodies for 1 h at room temperature. AlexaFluor 647-conjugated anti-guinea pig secondary antibodies (Thermo Fisher) were used. Coverslips were then washed 3 times and incubated LipidTOX green (1:1000, Thermo Fisher) and DAPI (10 μg/mL) in PBS for 45 min at room temperature. Slides were mounted with SlowFade Gold (ThermoFisher). Imaging was performed with the DeltaVision Core Image Restoration Microscope (GE Healthcare).
7
Lipid Accumulation Imaging Protocol
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LipidTOX Green was used according to the manufacturer’s instructions (ThermoFisher). Briefly, cells grown in 12-well plates were fixed in 3.5% (v/v) formaldehyde and washed extensively with PBS. The stain was used at a 1:1000 dilution in PBS. Cells were stained for 2 hours and kept in PBS at 4 °C until imaging. At least 5 random fields/well were captured on an inverted microscope (Olympus IX73) using identical settings and exposure times. Representative images are displayed in the panels. The fluorescence in each image was quantified in ImageJ and corrected for cell numbers. The mean fluorescence intensities -/+ SD across all images within each experimental group are provided in the figures.
8
Lipid Visualization in Fixed Cells
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Indicated cells were fixed in 4 %w/v paraformaldehyde and stained with LipidTox Green (ThermoFisher Scientific) according to manufacturer’s instructions, and 5 ng/mL Hoecsht 3342 (Cell Signalling) for 15 minutes prior to imaging using a Leica microscope system. Images were processing using Fiji.
9
Multilineage Differentiation of muBM-MSCs
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muBM-MSCs were seeded onto Primaria™ 6-well plates and cultured in differentiating media (2 mL) (StemPro™ Osteogenesis Differentiation Kit, Adipogenesis Differentiation Kit, Chondrogenesis Differentiation Kit, Life technologies). For osteocyte and adipocyte differentiation cells were seeded at 80,000 per well, for chondrocyte differentiation cells were plated as 5 μL droplets of cell solution at a concentration of 160,000,000 cells /mL. Cells were cultured for 2–4 weeks before fixing with 4% paraformaldehyde (2 mL) (Sigma-Aldrich). Cultures were stained with either; 1% Lipidtoxgreen (2 mL) (Thermo Fisher Scientific, Waltham, Massachusetts), 2% Alizarin Red (2 mL) (Abcam, Milton, UK), 1% Alcian Blue (2 mL) (Sigma-Aldrich). Cultures rinsed with PBS (3x, 2 mL) then imaged at 100x using an Axiovert 35 inverted phase contrast and fluorescence microscope (Zeiss, Jena, Germany).
10
Multilineage Differentiation of MSCs
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Cells were plated in MSC media (Lonza) at 80 % confluence into six-well dishes and incubated for 8–12 hours to allow cell attachment. After the cells were attached, the media were changed to the respective differentiation cocktails ± 100 nM tazarotene (Sigma) for 14 days in vitro (DIV). Commercially available differentiation cocktails used were StemPro® Adipogenesis, Osteogenesis, and Chondrogenesis Differentiation Kits (Life Technologies). After 14 days, cells were fixed with 4 % paraformaldehyde for 30 minutes and stained for lineage specific markers. Akaline phosphatase activity in osteoblasts was revealed using Fast Blue RR (Sigma). Adipocytes were stained for lipid accumulation with LipidTOX-Green (Life Technologies). After fixation, cells were incubated with PBS containing LipidTOX for 1 hour and then imaged. Chondrocytes were examined for aggrecan accumulation using the immunofluorescence protocol already described.
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