Visualization of mitochondria and lipid droplets in live cells was achieved with MitoTracker Red and HCS LipidTOX™ Green Neutral Lipid Stain, respectively (Life Technologies). Imaging of stained mitochondria and GFP activity was carried out on adipocytes 6 days after treatment with DHA or OA and the effect of bacterial infection was monitored 20 h after addition of LPS. Immunofluorescence was carried out on PFA fixed (4% paraformaldehyde in 1 × PBS) and saponin (0.2% saponin in 1 × PBS) permeabilized cells. FATP1 mediated uptake of fatty acids was investigated using a mouse monoclonal antibody (Diluted 100× as described in Sanchez-Gurmaches, et al. [66 (link)], R&D Systems, MN, USA) and oxidative stress was studied using a rabbit polyclonal antibody against iNOS (Diluted 200×, as described in Ebbesson, et al. [67 (link)], Thermo Scientific, IL, USA). Micrographs were captured and analysed using a Zeiss Axiovision Z1 microscope and Zeiss Axiovision software, respectively (Carl Zeiss Microimaging GmbH, Göttingen, Germany).
Hcs lipidtox green neutral lipid stain
Manufactured by Thermo Fisher Scientific
Sourced in United States, Poland
The HCS LipidTOX™ Green Neutral Lipid Stain is a fluorescent dye used to detect and quantify neutral lipids in cells. It emits green fluorescence when bound to neutral lipids, allowing for visualization and analysis of lipid content in samples.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 510, by Zeiss (4 mentions)
Hoechst 33342, by Thermo Fisher Scientific (4 mentions)
Lysotracker red dnd 99, by Thermo Fisher Scientific (2 mentions)
Prolong diamond antifade mountant with dapi, by Thermo Fisher Scientific (2 mentions)
Fibronectin, by Merck Group (2 mentions)
Observer z1 confocal spinning disc v 2, by Zeiss (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Lsm800 confocal microscope, by Zeiss (2 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (2 mentions)
Mitotracker red, by Thermo Fisher Scientific (1 mentions)
Axiovision software, by Zeiss (1 mentions)
Axiovision z1 microscope, by Zeiss (1 mentions)
Zen 2010, by Zeiss (1 mentions)
Mitotracker deep red fm, by Thermo Fisher Scientific (1 mentions)
2 nbdg, by Thermo Fisher Scientific (1 mentions)
Mitosox red, by Thermo Fisher Scientific (1 mentions)
Facscanto 2, by BD (1 mentions)
Hanks balanced salt solution (hbss), by Merck Group (1 mentions)
Fluoromount g, by Southern Biotech (1 mentions)
Protease k, by Merck Group (1 mentions)
29 protocols using hcs lipidtox green neutral lipid stain
1
Visualizing Mitochondria and Lipid Droplets
2
Multimodal Adipocyte Imaging Protocol
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For UCP1 staining, adherent adipocytes were fixed with 5% paraformaldehyde for 10 min at room temperature (RT), followed by permeabilization with 1% Triton X-100 for another 10 min at RT. Cells were then blocked using Blocking Buffer 1 (2% BSA and 2.5% goat serum) and incubated for 1 h at RT. Next, plates were incubated with primary antibody for 16 h at 4 °C. Primary antibody containing UCP1 (Sigma U6382, 1:100) was diluted in Blocking Buffer 2 (2% BSA).
For MitoTracker staining, 25 nM of MitoTracker was added onto live cells and incubated at 37 °C for 30 min. After staining, cells were washed 2 times with fresh media and then fixed with 5% paraformaldehyde for 10 min at RT.
For both types of staining, plates were washed 3 times with 1X PBS and secondary antibody was added. Secondary antibody containing Hoechst (1:6000) for nuclei staining, Alexa Fluor 647 goat Anti-Rabbit (1:1000) (for UCP1 staining only), and HCS LipidTOX Green Neutral Lipid Stain (Life Technologies, Carlsbad, CA, USA, 1:800) were diluted in Blocking Buffer 2. After 1 h of incubation at room temperature, plates were washed 3 times with 1X PBS and left in 1X PBS to be imaged.
For MitoTracker staining, 25 nM of MitoTracker was added onto live cells and incubated at 37 °C for 30 min. After staining, cells were washed 2 times with fresh media and then fixed with 5% paraformaldehyde for 10 min at RT.
For both types of staining, plates were washed 3 times with 1X PBS and secondary antibody was added. Secondary antibody containing Hoechst (1:6000) for nuclei staining, Alexa Fluor 647 goat Anti-Rabbit (1:1000) (for UCP1 staining only), and HCS LipidTOX Green Neutral Lipid Stain (Life Technologies, Carlsbad, CA, USA, 1:800) were diluted in Blocking Buffer 2. After 1 h of incubation at room temperature, plates were washed 3 times with 1X PBS and left in 1X PBS to be imaged.
3
Fluorescence Microscopy for Lipid Droplet Analysis
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For fluorescence microscopy, cells were grown on a coverslip to 50% confluency. For lysosomal staining, cells were incubated with 0.5 μmol/L Lysotracker Red DND-99 (Life Technologies; L7528) in full medium for 45 minutes before fixation. Cells were fixed with 4% PFA in phosphate-buffered saline for 10 minutes at room temperature. For determination of neutral lipids enclosed in cytosolic lipid droplets, cells were incubated with HCS LipidTOX Green neutral lipid stain (Life Technologies; H34475) according to manufacturer’s protocol. For imaging, cells were mounted with Vectaschield DAPI stain (Vector Laboratories, Burlingame, CA; H-1000-10) according to manufacturer’s protocol for nuclear staining. Cells were imaged at room temperature on a Zeiss (Jena, Germany) Observer Z1 brightfield microscope equipped with ApoTome 2 and a 63× (W) objective lens (APO DIC III numerical aperture 1.2) and acquired using Zeiss software Zen 2010. Laser lines used in this study were 405, 488, and 568 nm. Red/green/blue and greyscale images were further processed with Zeiss software Zen 2010. In addition, HepG2 cells and HLCs were used to assess cellular and surface LDL receptor protein expression levels with Western blot and flow cytometry and LDL uptake with Dylight labeled LDL particles in combination with flow cytometry.24
4
Quantifying Intracellular Neutral Lipids
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For assessment of neutral lipid formation, cells were washed in PBS, fixed with 4% paraformaldehyde for 30 min, washed in distilled water, and then briefly incubated in 60% isopropanol. Following equilibration, cells were stained with a working solution (60%) of Oil Red O for 20 min. An Oil Red O stock solution was prepared by dissolving 0.35% w/v Oil Red O in 100 mL 100% isopropanol. HCS LipidTOX™ Green Neutral Lipid Stain (Invitrogen; Cat. #H34475) was employed as a second staining cocktail for visualization of intracellular neutral lipids. Formaldehyde fixation and preparing/using the labeling solution was carried out as per the manufacturer’s instructions.
5
Metabolic Profiling of Activated CD4+ T Cells
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CD4+ TEMs were activated with plate-bound anti-CD3 or NIB1412 for 48 hours. For the quantification of mitochondria, cells were stained with MitoTracker® Deep Red FM (M22426; Molecular Probes) at 20 nM during the last 30 minutes of treatment. Cells were washed with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde and stained with HCS LipidTOX™ Green Neutral Lipid Stain (H34475; Invitrogen) at 1:500. To quantify mitochondrial superoxide production, cells were incubated with MitoSOX™ Red (Cat No. M36008; Invitrogen) at 37°C for 10 minutes, washed and fixed in 2% paraformaldehyde. MitoSOX Red was excited at 488 nm and fluorescence emission at 575 nm was measured.
For the determination of glucose uptake and cell surface expression of glucose transporters, cells were incubated with 2-NBDG (N13195; Molecular Probes) for 30 minutes, washed three times, and then stained with anti-Glut1-PE (MAB1418; R&D Systems) for 20 minutes. Cells were then washed and fixed with 4% paraformaldehyde.
Staining and incubations were performed at 37°C. Untreated cells were used as controls. Fluorescent signals from cells were acquired on BD FACS Canto II flow cytometer and data were analyzed using Cyflogic software v. 1.2.1.
For the determination of glucose uptake and cell surface expression of glucose transporters, cells were incubated with 2-NBDG (N13195; Molecular Probes) for 30 minutes, washed three times, and then stained with anti-Glut1-PE (MAB1418; R&D Systems) for 20 minutes. Cells were then washed and fixed with 4% paraformaldehyde.
Staining and incubations were performed at 37°C. Untreated cells were used as controls. Fluorescent signals from cells were acquired on BD FACS Canto II flow cytometer and data were analyzed using Cyflogic software v. 1.2.1.
6
Quantifying Neutral Lipid Droplets in HepG2 Cells
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The accumulation of neutral lipid droplets was analyzed using an HCS LipidTOX™ Green Neutral Lipid Stain (Invitrogen Life Technologies, Warsaw, Poland) for cellular imaging. Staining was performed in accordance with the manufacturer’s instructions. Briefly, all treated and untreated HepG2 cells were fixed with 4% paraformaldehyde for 40 min at room temperature, washed three times with HBSS (Sigma Aldrich, Poznań, Poland), and labeled with LipidTOX™ Green for 20 min at room temperature. Subsequently, the nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) using the ProLong™ Diamond Antifade Mountant with DAPI (Invitrogen Life Technologies, Warsaw, Poland). Photomicrographs were captured using a confocal microscope (Observer Z1 Confocal Spinning Disc V.2 Zeiss with a live imaging chamber). The obtained photomicrographs were then merged and analyzed using the ImageJ software (Bethesda, MD, USA).
7
Quantifying mT/mG and Leptin-Luciferase Adipocytes
8
Comprehensive Lipid Analysis Protocol
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FFA fluorometric assay kit (Cayman, item no.700310); Lipofectamine 3000 (Invitrogen, L3000–015); Norepinephrine (Sigma, A9512); HCS LipidTOX™ Deep Red Neutral Lipid Stain (Invitrogen™, H34477); HCS LipidTOX™ Green Neutral Lipid Stain (Invitrogen™, H34477); Trypsin Platinum (Promega, VA9000); Protease K (Sigma, P2308); Oleic acid (Sigma, O1383); BSA (Sigma, A7030); FFA-free BSA (Millipore, Code82-002-4); Trypsin inhibitor (Worthington, LS003571); Protease inhibitor cocktail (100X) (Thermo scientific, 1861279); Free glycerol reagent (Sigma, F6428–40ML); Propargyl choline (Cayman, 25870); BTTAA (Click chemistry tools, 1236–100); CalFluor 647 Azide(Click chemistry tools, 1272–1); Phosphatidylcholine Assay Kit (Colorimetric/Fluorometric) (Abcam , ab83377); Phosphatidylethanolamine Assay Kit (Fluorometric) (Sigma, MAK361).
9
Lipid Droplet Imaging in ASCs
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Neutral lipid droplets in all cultured ASCs cells were analysed using the fluorescent HCS LipidTOX™ Green Neutral Lipid Stain (Invitrogen Life Technologies, Warsaw, Poland) for cellular imaging. Staining was performed following the protocol of the manufacturer as described elsewhere [37 (link)]. ASCs cells were fixed in 4% paraformaldehyde for 40 min prior to staining with LipidTOX™ Green during 20 min at room temperature. After three HBSS washes, the nuclei were counterstained with DAPI using the ProLong™ Diamond Antifade Mountant with DAPI (Invitrogen Life Technologies, Warsaw, Poland). Photomicrographs were captured using a confocal microscope (Observer Z1 Confocal Spinning Disc V.2, Zeiss with a live imaging chamber). The obtained photomicrographs were then merged and analysed using the ImageJ software (Bethesda, MD, USA).
10
Multimodal Microscopy Imaging Techniques
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Cells were counted and seeded on a glass-bottom dish (3.5-cm diameter, No. 1.5 MatTek, Ashland, MA, USA) coated with fibronectin (Millipore) 1 day before imaging as previously described [36 (link)]. Mitochondria were labeled with 100 nM MitoTracker Green FM/Orange CMTMRos/Deep Red FM (Invitrogen, Eugene, OR, USA) for 3 min, and then washed and incubated in Live Cell Imaging Solution at 37 °C for 10 min prior to imaging. Lipid droplets were labeled with HCS LipidTOX Green Neutral Lipid Stain (Invitrogen) as recommended by the manufacturer. Furthermore, 100 mM BODIPY 558/568 C12 (C12, Invitrogen, Eugene, OR, USA) was prepared, mixed with 100 mM PA at a 1:2000 ratio and then added to BM instead of PA for visualization of PA incorporation into mitochondria and lipid droplets. All pictures were taken with live cells using spinning disk confocal microscopy (SDCM).
Colocalization of DRP1 and TOM20 z-stack images are taken with Zeiss LSM510. Cells were fixed in 4% paraformaldehyde for 20 min, washed with PBS, permeabilized with 0.3% NP40, 0.05% Triton-X100 in PBS for 3 min and incubated with corresponding primary antibodies to TOM20 and DRP1 overnight at 4 °C followed by Alexa 488- and Alexa 647-labelled secondary antibodies next day for 60 min at room temperature.
Colocalization of DRP1 and TOM20 z-stack images are taken with Zeiss LSM510. Cells were fixed in 4% paraformaldehyde for 20 min, washed with PBS, permeabilized with 0.3% NP40, 0.05% Triton-X100 in PBS for 3 min and incubated with corresponding primary antibodies to TOM20 and DRP1 overnight at 4 °C followed by Alexa 488- and Alexa 647-labelled secondary antibodies next day for 60 min at room temperature.
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