Fibroblasts were cultured in adipogenic differentiation medium (Lonza, Basel, Switzerland) for 2 weeks and stained with 1:1,000 HCS LipidTOX™ Deep Red Neutral Lipid Stain (Thermo Fisher Scientific, Waltham, Massachusetts). Images were taken using a Scan R high content screening station (Olympus, Tokyo, Japan). The percentage of differentiated fibroblasts was quantified using Scan R analysis software version 3.1.1. Fibroblasts were cultured in chondrogenic differentiation medium (Promocell, Heidelberg, Germany) for 2 to 3 weeks and time in days was measured until three-dimensional cartilage-like structure formed (Supplementary Figure S7 ).
Hcs lipidtox deep red neutral lipid stain
Manufactured by Thermo Fisher Scientific
Sourced in United States
The HCS LipidTOX™ Deep Red Neutral Lipid Stain is a fluorescent dye used for the detection and visualization of neutral lipids in cells. It emits a deep red fluorescence when bound to neutral lipids.
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Lab products found in correlation
Bodipy 493 503, by Thermo Fisher Scientific (6 mentions)
Axio imager, by Zeiss (4 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (3 mentions)
Pegfp n3, by Takara Bio (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488 conjugated goat anti rabbit igg, by Thermo Fisher Scientific (2 mentions)
Adipogenic differentiation medium, by Lonza (1 mentions)
Chondrogenic differentiation medium, by PromoCell (1 mentions)
Scanr high content screening station, by Olympus (1 mentions)
Bodipy fl c12, by Thermo Fisher Scientific (1 mentions)
Bodipy 493 503 dye, by Thermo Fisher Scientific (1 mentions)
Cellmask orange, by Thermo Fisher Scientific (1 mentions)
D eclipse c1 confocal microscope, by Nikon (1 mentions)
Mitotracker orange cmtmros, by Thermo Fisher Scientific (1 mentions)
Sp8x confocal microscope, by Leica camera (1 mentions)
Hrp conjugated anti mouse igg antibody, by Thermo Fisher Scientific (1 mentions)
Hoechst 33258, by Thermo Fisher Scientific (1 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions)
31 protocols using hcs lipidtox deep red neutral lipid stain
1
Adipogenic and Chondrogenic Differentiation
2
Lipid Droplet Staining in Cancer Cells
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Two days before the experiment, cells were plated on Geltrex-coated coverslips. To stain the LDs of WiDr cells, the cells were incubated with BODIPY FL C12 (Thermo Fisher Scientific) for 8 h. After washing the cells with PBS, they were fixed in 4% paraformaldehyde. For staining the LDs of PANC-1 cells, the cells were fixed with 4% paraformaldehyde and then stained with HCS LipidTOX Deep Red neutral lipid stain (Thermo Fisher Scientific) according to the manufacturer’s protocol. Cells were mounted using glycerol/PBS solution. Fluorescence images were acquired using a FLUOVIEW FV10i confocal laser-scanning microscope system (Olympus, Tokyo, Japan).
3
Differentiating Primary Mouse Adipocytes
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Primary SVF cells were isolated from BAT and ingWAT of 3-4 week old mice. Cells were maintained in Dulbecco’s modified Eagle’s medium (high glucose) containing 10% bovine serum at 37°C in a 5% CO2 incubator.Cells were allowed to reach confluency, and treated with induction media supplemented with 2% FBS, 20 nM insulin, 1 nM triiodothyronine (T3), 0.125 mM indomethacin, 5 μM dexamethasone, 0.5 mM IBMX, and 1 μM Rosiglitazone for 2 days. Then, cells were maintained in differentiation media supplemented with 2% FBS, 20 nM insulin, 1 nM T3, and 1 μM Rosiglitazone for 6 more days. During differentiation, media was changed every other day. Staining for neutral lipid was done using HCS LipidTOX™ Deep Red Neutral Lipid Stain (ThermoFisher Scientific, H34477).
4
Characterization of NRB Protein Localization
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To characterize NRBMC009 and NRBZLW0047 localization, each were diluted in HL3 medium at concentrations of 500 nM and 1 μM, respectively, and were added to the dissected larva and images acquired after 15 min. For NRBMC009 colocalization studies, ER-Tracker Red 2 μM (BODIPY TR Glibenclamide), MitoTracker Orange CMTMRos 1 μM, LysoTracker Deep Red 2 μM, HCS LipidTOX Deep Red Neutral Lipid Stain 1:100, or CellMask Orange 1 μM (all by Thermo Fisher, respectively #E34250, #M7510, #L12492, #H34477, and #10045) were added, together with NRBMC009 at 500 nM. To verify NRBZLW0047 colocalization with other organelles, it was added on dissected larvae expressing GFP-tagged proteins (Hneu-GFP, Mito-GFP, Lamp-GFP, and mCD8-GFP), or together with BODIPY 493/503 dye 10 μg/ml (#D3922, Thermo Fisher). In order to test tissue autofluorescence, dissected larvae were imaged in HL3 medium without fluorescent probes with identical laser settings of labeled tissues (S1 Fig ). Whole larvae images and magnifications were acquired using a Zeiss LSM800 Axio Observer Z1 inverted microscope equipped with a Zeiss Plan-Apochromat 5x/0.15 ph1 or 40x/0.95 objectives, all other images were acquired with a Nikon D-Eclipse C1 confocal microscope equipped with a Nikon Plan Apo 60×/1.40 or a Nikon Plan Apo 40x/1.0 oil immersion objectives.
5
Adipogenesis Evaluation via Lipid Staining
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After 7 days of adipogenic differentiation, cells were fixed with 4% (vol/vol) paraformaldehyde (PFA) for 15 min at RT and then incubated with 0.5 µM BODIPY® 493/503 (Thermo Fisher Scientific) diluted in PBS or HCS LipidTOX™ Deep Red Neutral Lipid Stain (Thermo Fisher Scientific) 1X diluted in PBS for 20 min at room temperature (RT) in the dark. HCS LipidTOX™ Deep Red Neutral Lipid Stain was used only for GCN5 knockdown experiment in ACM CStCs transduced with GFP‐tagged lentiviral particles, in order to avoid spectral overlap between GFP and BODIPY® 493/503. Nuclei were counterstained with DAPI (Thermo Fisher Scientific). Immunofluorescence images were acquired using a Leica SP8‐X confocal microscope, and the fluorescence integrated intensity quantified using Fiji/Image J Software.
6
Acquisition of HCV Antiviral Compounds
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DCV (BMS-790052) and DNV (RG7227) were purchased from Selleck Chemicals, (Houston, TX, USA). LDV (GS-5885) was purchased from MedChem Express (Monmouth Junction, NJ, USA). SOF (GS-7977) was purchased from Acme Bioscience (Palo Alto, CA, USA). Hoechst-33258 and BODIPY 493/503 were purchased from Invitrogen (Waltham, MA, USA), and HCS LipidTOX Deep Red Neutral Lipid Stain was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Cyclosporin A (CsA—30024) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Mouse monoclonal primary antibody 9E10, specific for NS5A, was provided by Brett Lindenbach (Yale University) [61 (link)]. Mouse monoclonal antibodies, specific for HCV core (C7-50), dsRNA (J2), and GAPDH (G-9), were purchased from Abcam (Waltham, MA, USA), Jena Bioscience (Jena, Germany), and Santa Cruz Biotechnology (Santa Cruz, CA, USA), respectively. Secondary antibodies Alexa 488-, 647- and HRP-conjugated anti-mouse IgG antibodies were purchased from Invitrogen and Santa Cruz Biotechnology, respectively.
7
Lipid Droplet Isolation and Analysis
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Fetal calf serum, high-glucose Dulbecco's Modified Eagle’s Medium, penicillin/streptomycin, Opti-MEM reduced serum medium, Lipofectamine 3000 transfection reagent, HCS LipidTOX Deep Red Neutral Lipid Stain, and BODIPY 493/503 were from Thermo Fisher Scientific. Oleate-bovine serum albumin complexes, primers, protease inhibitor cocktail, and Tween-20 were from Sigma-Aldrich. Tris-glycine SDS-PAGE gels were prepared in-house. Immobilon Western chemiluminescent HRP substrate and nitrocellulose membranes were from Millipore. Skim milk powder was from Fonterra. The Lipid Droplet Isolation Kit was from Cell Biolabs Inc.
8
Lipid Droplet Quantification in Mtb-Infected MH-S Cells
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Adherent MH-S cells were grown on coverslips (Fisherbrand) placed in 6-well plates (Corning). Following Mtb infection, cells were fixed in 4% formaldehyde, washed and then stained with PBS solution of HCS LipidTOX Deep Red Neutral Lipid stain (ThermoFisher Scientific), followed by 300 nM DAPI nuclear stain solution (ThermoFisher Scientific). coverslips were then washed three times with PBS and then mounted on Super frost/Plus microscope slides using Molecular Probes Slowfade Light antifade medium. Nikon A1RS confocal microscope was used to acquire images and quantification of signal intensity was performed using ImageJ software and Nikon imaging software, Nikon Elements 4.5.
9
Lipid Profiling of Tumor-Associated Macrophages
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Single-cell suspensions from surgically resected CLM tissues from four patients were obtained as described in the previous paragraph. To assess expression of lipid transporters (ABCA1 and ABCG1), ApoE, and CD36, the following antibodies were included in the cocktail: anti-ABCA1 (Invitrogen; rabbit polyclonal), anti-ABCG1 (Novus Biologicals; rabbit polyclonal), anti-ApoE (BioLegend; clone E6D7), and anti-CD36 (BioLegend; clone 5–271). Detection of ABCG1 and ApoE was performed by intracellular staining with BD Cytofix/Cytoperm Solution (BD Biosciences). Cells were acquired on a FACSAria III (BD Biosciences) following the gating strategy shown in Fig. S2 .
For lipid staining, cells were stained with the same cocktail used for cell sorting and then incubated with HCS LipidTOX Deep Red Neutral Lipid Stain (ThermoFisher; dilution 1:200) in PBS for 30 min at room temperature. Cells were immediately washed with PBS and acquired by flow cytometer. Lipid content of L-TAMs and S-TAMs was evaluated as mean fluorescence intensity (MFI) of the dye.
To test lipid uptake, cells were incubated with 0.8 µM of the fluorescent dye BODIPY 558/568 C12 (ThermoFisher) in PBS containing 0.1% BSA (fatty acid–free) for 1 min at 37°C. Cells were immediately washed with cold PBS containing 0.2% BSA and acquired by flow cytometer. Lipid uptake of L-TAMs and S-TAMs was evaluated as MFI of the dye.
For lipid staining, cells were stained with the same cocktail used for cell sorting and then incubated with HCS LipidTOX Deep Red Neutral Lipid Stain (ThermoFisher; dilution 1:200) in PBS for 30 min at room temperature. Cells were immediately washed with PBS and acquired by flow cytometer. Lipid content of L-TAMs and S-TAMs was evaluated as mean fluorescence intensity (MFI) of the dye.
To test lipid uptake, cells were incubated with 0.8 µM of the fluorescent dye BODIPY 558/568 C12 (ThermoFisher) in PBS containing 0.1% BSA (fatty acid–free) for 1 min at 37°C. Cells were immediately washed with cold PBS containing 0.2% BSA and acquired by flow cytometer. Lipid uptake of L-TAMs and S-TAMs was evaluated as MFI of the dye.
10
Intracellular Calcium and Cytoskeleton Changes in nsPEF-Treated Cells
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Cells were trypsinized and seeded on microscopic cover glasses (24 × 24 mm, Carl Roth GmbH, Germany) in 35 mm Petri dishes (Nunc, Biokom, Janki, Poland). Cells were left to adhere overnight. Cells were then exposed to nsPEF or nsPEF with 1 mM Ca2+ or 2 mM Ca2+, and left for 24 h. After this time, cells were fixed 10 min in 4% paraformaldehyde (Polysciences, Inc., Bergstrasse, Germany) and washed in PBS (BioShop, EPRO, Poland). Cytoskeleton reorganization was determined by immunofluorescent cells’ labeling of F-actin with Alexa Fluor®546 Phalloidin (Thermo Fisher, distributor: Life Technologies, Warsaw, Poland), and intracellular calcium was determined by 4 µM Fluo-4 (λExc.494/λEm.506 nm, F14201, Thermo Fisher Scientific), lipid droplets by HCS LipidTOX™ Deep Red Neutral Lipid Stain (H34477, Thermo Fisher Scientific). FluorshieldTM with DAPI (4,6-diamidino-2-phenylindole) was used to visualize the nuclei and to mount the cells, and was excited by a 405 nm-excitation channel. The samples were studied on the Olympus FluoView FV1000 confocal laser scanning microscope (Olympus, Tokyo, Japan).
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