The following chemicals were used at the indicated concentrations: 20, 50, and 100 ng/ml Doxycycline (Sigma-Aldrich, D5207), 50 nM CHIR (Selleckchem, S2745), 3 µM PIM447 (Selleckchem, S7985), 4 µM GW6471 (Selleckchem, S2798), 100 µM Etomoxir (Selleckchem, S8244), 20 µg/ml cycloheximide (Selleckchem, S7418), 1:1000 LipidSpot488 (Biotium, 70065) or LipidSpot610 (Biotium, 70069), 167 nM SyTOX Green Nucleic Acid Stain (Fisher Scientific, S7020), 25 mM Glucose (Thermo Scientific, A24940-01), 10% Dialyzed FBS (Gibco, A33820-01).
Lipidspot 488
Manufactured by Biotium
Sourced in United States
LipidSpot 488 is a fluorescent dye used for labeling and visualizing lipids in biological samples. It selectively binds to lipids, allowing for their detection and analysis under fluorescent microscopy.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Lipidspot 610, by Biotium (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Cycloheximide (chx), by Selleck Chemicals (1 mentions)
Doxycycline, by Merck Group (1 mentions)
Etomoxir, by Selleck Chemicals (1 mentions)
Gw6471, by Selleck Chemicals (1 mentions)
Sytox green nucleic acid stain, by Thermo Fisher Scientific (1 mentions)
Dialyzed fbs, by Thermo Fisher Scientific (1 mentions)
Pim447, by Selleck Chemicals (1 mentions)
Dfc7000 t camera, by Leica (1 mentions)
Las x software, by Leica (1 mentions)
Dm lb 11888011, by Leica (1 mentions)
Hibernate a, by Transnetyx (1 mentions)
Sybr green, by Thermo Fisher Scientific (1 mentions)
Lab tek 8 well chambered coverglass, by Thermo Fisher Scientific (1 mentions)
Hepes, by Thermo Fisher Scientific (1 mentions)
Neon transfection system, by Thermo Fisher Scientific (1 mentions)
12 protocols using lipidspot 488
1
Multiparametric Lipid Imaging Assay
2
Immunofluorescence Imaging of Cellular Organelles
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Cells were plated in six-well plates containing microscope coverslips and treated as indicated. After treatment, cells were fixed with 4% formaldehyde for 20 min. and kept in a blocking solution (5% NGS and 0.3% Triton X-100 in PBS) for 60 min. Then, cells were incubated with anti-Catalase (CST, 12980), anti-PPARα (LifeSpan BioSciences, LS-B46), anti-Tom20 (CST, 42406) antibodies for 60 min. Following primary antibody incubation, cells were washed with 1X DPBS and incubated in secondary antibodies (Alexa Fluor 568 goat anti-mouse and Alexa Fluor 488 goat anti-rabbit, 1:500 dilution) for 60 min. Finally, cells were mounted on glass slides with mounting media (CST, Prolong® Gold, 8961) containing DAPI. Images were taken at 60× magnification using a fluorescent microscope. For LD imaging, cells were seeded and fixed in 4% formaldehyde as described above followed by 30 min incubation in 1:1000 diluted LipidSpot 488 or LipidSpot610 (Biotium). After staining, cells were washed in 1X DPBS and mounted onto slides.
3
Lipid Droplet Staining in Fungal Mycelia
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Strains were grown on glass slides with a central depression containing 130 µL M2 medium or linoleic acid-containing M2 medium (0.8 mM) for one day under standard conditions. For the staining of lipid droplets, grown mycelium was incubated with LipidSpot™ 488 (Biotium, Fremont, CA, USA; 70065) for 15 min. Fluorescence microscopic analyses were performed with a fluorescence microscope (DM LB/11888011, Leica, Wetzlar, Germany) and a DFC7000 T camera; image processing was conducted with the corresponding LAS X software from Leica (Leica Microsystems GmbH, Wetzlar, Germany).
4
Cell Culture and Imaging Techniques
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COS-7, U2-OS, and HeLa cells (Cell Culture Facility, UC-Berkeley) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco 31053-028) supplemented with 10% fetal bovine serum (Corning), 1× GlutaMAX Supplement, and 1× non-essential amino acids, at 37 °C and 5% CO2. For live-cell experiments, cells were cultured in Lab-Tek 8-well chambered coverglass (ThermoFisher), and transiently transfected with the above plasmids, either alone or in combination. Transfection was performed using Lipofectamine 3000 (ThermoFisher) or the Neon Transfection System (ThermoFisher), following the manufacturers’ instructions. For live-cell staining of the plasma membrane, lipid droplet, and DNA, wheat germ agglutinin (WGA) CF532 (300–500×, 29064, Biotium), LipidSpot488 (300–1000×, 70065, Biotium), and SYBR Green (15,000–100,000×, S7536, ThermoFisher) were added to the medium for 30 min at 37 °C and washed three times with DMEM before imaging. Imaging buffer was the regular culture medium with the addition of 25 mM HEPES at pH 7.4 (15630106, Gibco) or a commercial buffer based on MOPS (Hibernate A, BrainBits), with similar results observed.
5
Neuroinflammation in APP/APOE Mouse Model
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Human APOE3-KI and APOE4-KI mice were cross-bred with mice overexpressing mutant human APP (J20 line) to generate J20/APOE4-KI and J20/APOE3-KI mice, as we reported previously13 . All mouse lines were maintained on a C57Bl/6J background. Sex- and age-matched wildtype mice were used as controls. Brains were collected from female J20/APOE4-KI and J20/APOE3-KI mice (n=3 for each group) at 13 months of age. Brain sections were collected (30μm) from paraformaldehyde-fixed right hemibrains on a sliding microtome fitted with a freezing stage as described previously. Free-floating 30 μm sections were washed three times in PBS followed by blocking with 10% donkey serum in PBS for 1 h. Sections were incubated in PBS with 5% donkey serum and Iba1 primary antibody (1:500; Wako 019–19741) for 72 h at 4 °C. After primary antibody incubation, sections were washed once in PBS and incubated in the following secondary antibody and lipid droplet dye for 2 hr at RT: donkey anti-rabbit 555 (1:500; Invitrogen) and LipidSpot 488 (1:1000; Biotium). Sections were incubated in DAPI (1:2000; Thermo Fisher) for 10 min, then washed three times with PBS. Sections were mounted on microscope slides with ProLong Glass mounting media. Imaging was performed with a ZEISS LSM 900 confocal microscope ZEN 3.0 (Blue Edition) software.
6
Lipid Staining of Squid Yolk Sac
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Squid were collected after 48 h of colonization and incubated at room temperature for 2 h in 1:1,000 of the lipid stain, lipidspot488 (Biotium). Afterward, the squid were washed in seawater, anesthetized in seawater containing 2% ethanol, and imaged using a Zeiss LSM 710 confocal microscope. Z-stack images were acquired and the area of the internal yolk sac measured using FIJI [85 (link)] from the sum slices of each Z-stack.
7
Lipid Droplet Staining in HT1080 Cells
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For staining of lipid droplets, HT1080 cells were plated in 35-mm glass bottom dishes (ibidi, μ-Dish 35-mm high Glass Bottom) and treated with 200 μM oleic acid overnight. Cells were then fixed for 10 minutes in 4% paraformaldehyde preheated to 37°C, washed in PBS (pH 7.4) and permeabilized with 0.2% saponin (Nacalai USA, San Diego, CA, USA) in PBS for 30 minutes at room temperature. Lipid droplets, F-actin, and nuclei were stained with LipidSpot 488 (Biotium, Fremont, CA, USA), Phalloidin-ATTO643 (ATTO-TEC, Siegen, Germany), and Hoechst 33342 (Thermo Fisher), respectively.
8
Visualizing Subcellular Lipid Droplets
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The subcellular distribution of cell nuclei and lipid droplets was examined by fluorescence. After 7 days of coculture, cells were fixed with 4% formaldehyde and permeabilized with 0.1% Triton X-100 in PBS. Unspecific binding sites were then blocked with 1% bovine serum albumin (Sigma Aldrich, Burlington, Massachusetts, USA). LipidSpot 488 (Biotium, Fremont, California, USA) was applied to visualize lipid droplets present inside the cells. Cell nuclei were visualized with Hoechst 33342 reagent (Invitrogen, Waltham, Massachusetts, USA). Then, coverslips were mounted with Dako fluorescent mounting medium (Agilent, Santa Clara, California, USA). For each condition, representative cells are shown. Experiments were conducted with three repetitions.
9
Multicolor Imaging of Cellular Organelles
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For fixed-cell experiments, COS-7 cells were plated in an 8-well LabTek chamber at ~30% confluency. After 24 h, cells were fixed using 3% paraformaldehyde and 0.1% glutaraldehyde in phosphate-buffered saline (PBS) followed by two washes with 0.1% sodium borohydride in PBS. Cells were blocked and permeabilized in a blocking buffer (3% bovine serum albumin with either 0.5% Triton X-100 or 0.02% saponin in PBS), followed by primary and secondary antibody labeling. Primary antibodies used were chicken anti-α-tubulin (ab89984, Abcam) for labeling of microtubules, rabbit anti-Nogo (ab47085, Abcam) for labeling of ER, mouse IgG1 anti-NPM1 (32-5200, ThermoFisher) for labeling of nucleoli, and mouse IgG2a anti-Tom20 (sc-17764, Santa Cruz Biotech) for labeling of mitochondria. Secondary antibodies (Jackson ImmunoResearch) were labeled via reaction with dye NHS esters: CF514 (92103, Biotium), CF568 (92131, Biotium), ATTO 532 (AD532-31, ATTO-TEC) and ATTO 542 (AD542-31, ATTO-TEC). After antibody staining, cells were incubated in LipidSpot488 (300–1000×, 70065, Biotium) and SYBR Gold (300–1000×, S11494, ThermoFisher) in PBS to stain lipid droplets and nuclear DNA for 30 min, and the sample was washed with PBS for 10 min × 3 times before imaging in PBS.
10
Lipid Dynamics in SARS-CoV-2 Infection
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Calu‐3 cells were suspended in MEM medium with 2% lipid depleted serum and seeded in the wells of a Lab‐TEK II 4‐well chamber or a 96‐well plate. For the treatment with 25HC, cells were treated with 5 μM 25HC for 16 h. Then, the cells were washed and stained with 1× LipidSpot 488 (Biotium) for 30 min. The signals were captured by fluorescence microscope. For the infection with live SARS‐CoV‐2 virus, Calu‐3 cells were infected with SARS‐CoV‐2 USA‐WA1/2020 at MOI = 2 for 1 h at 37°C. Then, cells were washed, and fresh medium was supplemented. Cells were stained with 1× LipidSpot 488 for 30 min after 24 h post‐infection. The fluorescence signal was captured by Incucyte S3.
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