Filipin 3

Manufactured by Cayman Chemical

Sourced in United States

Filipin III is a fluorescent polyene compound that binds to cholesterol in biological membranes. It can be used as a stain for the detection and localization of cholesterol in cells and tissues.

Automatically generated - may contain errors

Lab products found in correlation

Spelling variants of this product

Filipin (12 citations)

33 protocols using filipin 3

Filipin III Staining of Fixed Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

Filipin III (Cat.70440, Cayman) was dissolved in ethanol to reach a final concentration of 5 mg/mL. Cells were fixed with 4% paraformaldehyde (PFA) and stained with 50 mg/mL Filipin III for 30-min at room temperature. The emission and excitation of Filipin III was at 340-380 and 385-470nm, respectively. The images were captured with Zeiss LSM980 confocal microscope using a 40x objective.

Lu M., He R., Li C., Liu Z., Chen Y., Yang B., Zhang X, & Yu B. (2023). Apolipoprotein E deficiency potentiates macrophage against Staphylococcus aureus in mice with osteomyelitis via regulating cholesterol metabolism. Frontiers in Cellular and Infection Microbiology, 13, 1187543.

+ Open protocol

+ Expand

Viral Cholesterol Content Quantification

Check if the same lab product or an alternative is used in the 5 most similar protocols

Viral stocks were treated with MβCD at various concentrations at 37 °C for 1 h followed by ultracentrifugation to remove the MβCD. The MβCD-treated virus supernatants were purified through a 20% sucrose cushion (wt/vol) prepared in TE buffer [10 mM Tris-HCl (pH 8.0), 1 mM EDTA] by centrifugation at 36,900 rpm for 1 h at 4 °C in a P70AT rotor (model CP100WX; Hitachi, Hitachinaka, Japan). The virion cholesterol content was determined using fluorescence intensity analysis. Briefly, 96-well plate wells were coated with 50 ng of the purified viruses in 50 mM sodium bicarbonate buffer (pH 9.6) and incubated at 4 °C overnight. Plates were washed three times with washing buffer (0.05% Tween-20 in PBS) and blocked with 5% powdered skim milk (BD Biosciences, Belford, MA) in PBS at 37 °C for 2 h. After washing, filipin III (Cayman Chemical, Ann Arbor, MI) was added in triplicate for 1 h in the dark. The plates were washed, and fluorescence intensity was measured with a SPARK 10M multimode microplate reader (TECAN, Männedorf, Switzerland). In parallel, the purified samples were used to infect PAM-pCD163 or Vero cells for 48 h, and the virus-infected cells were independently subjected to FACS analysis and virus titration to determine PRRSV or PEDV infection as described above.

Jeon J.H, & Lee C. (2017). Cellular cholesterol is required for porcine nidovirus infection. Archives of Virology, 162(12), 3753-3767.

+ Open protocol

+ Expand

Filipin III Staining for Cellular Cholesterol

Check if the same lab product or an alternative is used in the 5 most similar protocols

The cholesterol in the tissue or cells was stained by the cholesterol‐binding compound Filipin III (Cayman) for 2 h at room temperature. Fluorescence signals was analysed by a microscope (Olympus). Quantification of the staining was analysed by Image J.

Hu J., Zhu Z., Chen Z., Yang Q., Liang W, & Ding G. (2022). Alteration in Rab11‐mediated endocytic trafficking of LDL receptor contributes to angiotensin II‐induced cholesterol accumulation and injury in podocytes. Cell Proliferation, 55(6), e13229.

+ Open protocol

+ Expand

Cholesterol Metabolism Regulation

Check if the same lab product or an alternative is used in the 5 most similar protocols

27-Hydroxycholesterol was provided by American Santa Cruz, and 27-OHC (10 mg) was completely dissolved in a suitable amount of ethanol, then divided equally in centrifuge tubes, with nitrogen gas blowing dry, and finally preserved at −80°C. The 27-OHC working solution contains 0.16% ethanol (v/v). Filipin III was from Cayman Chemical (#70440, Ann Arbor, MI, United States). Primary antibodies for Western blot included mouse anti-ABCA1 (Abcam, ab66217), rabbit anti-ABCG1 (Abcam, ab52617), mouse anti-Caveolin-1 (Abcam, ab17052), rabbit anti-Apolipoprotein E (Abcam, ab52607), mouse anti-LDLR (Millipore, MABS26), rabbit anti-ACAT1 (Abcam, ab168342), rabbit anti-CYP46A1 (Sigma–Aldrich, SAB2100523), mouse anti-KDEL (Santa Cruz, sc-58774), rabbit anti-flotillin (Abcam, ab41927), rabbit anti-SR-B1 (Abcam, 52629), mouse anti- N Cadherin (Abcam, ab98952), mouse anti-Aβ (Abcam, ab126649), and rabbit anti-LRP1 (Abcam, ab92544). Goat anti-mouse (#14709) and rabbit (#7074) biotinylated secondary antibodies were from Cell Signaling Technology. Antibodies for immunofluorescence included anti-CYP46A1 (Abcam, ab82814), goat anti-mouse (Abcam, ab150120), and goat anti-rabbit (Abcam, ab96899).

Wang Y., Zhang X., Wang T., Liu W., Wang L., Hao L., Ju M, & Xiao R. (2020). 27-Hydroxycholesterol Promotes the Transfer of Astrocyte-Derived Cholesterol to Neurons in Co-cultured SH-SY5Y Cells and C6 Cells. Frontiers in Cell and Developmental Biology, 8, 580599.

+ Open protocol

+ Expand

Quantifying Lysosomal Cholesterol Accumulation

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

Similar to immunostaining, coverslips were fixed in 4% PFA for 20 min at room temperature first. They were treated with 0.05 mg/mL Filipin III (Cayman Chemical) in PBS with 10% FBS for 2 h at room temperature. Cells were incubated in primary antibody, anti-LAMP-1, with 5% BSA in PBS overnight at 4°C. Then cells were processed for secondary antibody incubation in 5% BSA for 45 min at room temperature and mounted on microscope slides. Triplicate coverslips were used to acquire images by using 60× objective of the fluorescence microscope. The colocalization images were analyzed using ImageJ software (Boutry et al., 2019 (link)). Specifically, the LAMP1-positive lysosomal area (first) and the soma of the cell (second area) were selected and added to ROI manager. We quantified the total fluorescent intensity of the first and second selection area under the blue channel (cholesterol stained). These areas represent the relative cholesterol level in the lysosome and soma, respectively. The ratio between two values is the proportion of cholesterol accumulation in the lysosome (Boutry et al., 2019 (link)). The proportion of cholesterol accumulated in lysosome was calculated by % = (filipin intensity in selected area of LAMP1⁺) / (filipin intensity in whole cell) × 100%.

Chai E., Chen Z., Mou Y., Thakur G., Zhan W, & Li X.J. (2023). Liver-X-receptor agonists rescue axonal degeneration in SPG11-deficient neurons via regulating cholesterol trafficking. Neurobiology of disease, 187, 106293.

+ Open protocol

+ Expand

Quantitative Imaging of Cellular Cholesterol

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cells grown on glass coverslips were fixed with 4% PFA and then stained with 5 μg/mL filipin-III (Cayman, USA) in PBS containing 1% FBS for 2 h to examine the intracellular distribution of free cholesterol. The stained cells were observed under an LSM 700 confocal microscope (Carl Zeiss, Germany). Signal intensities of plasma membranes were quantified using Image J software (http://imagej.nih.gov/ij/).

Kim T.Y., Jeon S., Ko M., Du Y.E., Son S.R., Jang D.S., Kim S, & Lee C.J. (2022). Lancemaside A from Codonopsis lanceolata: Studies on Antiviral Activity and Mechanism of Action against SARS-CoV-2 and Its Variants of Concern. Antimicrobial Agents and Chemotherapy, 66(12), e01201-22.

+ Open protocol

+ Expand

ARV Protein Immunofluorescence Protocol

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

Methyl-β-cyclodextrin (MβCD), cholesterol, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide (MTT), 4′,6-diamidino-2-phenylindole (DAPI) and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (Shanghai, China). Filipin III was purchased from Cayman (Michigan, United States). A mouse monoclonal antibody against σC of ARV and polyclonal antibodies against μNS and p10 of ARV were prepared by our laboratory (Duan et al., 2015 (link); Niu et al., 2019 (link)). A rabbit monoclonal antibody against Caveolin-1 was purchased from Cell Signaling Technology (Massachusetts, United States). A mouse monoclonal antibody against β-Actin and secondary antibodies, including fluorescein (FITC)-conjugated goat anti-mouse/rabbit IgG, horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG, and cholera toxin subunit B (CTB) conjugated to Alexa Fluor^® 594 (CTB- Alexa Fluor 594), were purchased from Sigma-Aldrich. RIPA lysis buffer, PMSF, and SDS-PAGE loading buffer were purchased from Beyotime Biotechnology (Shanghai, China). The chemiluminescent substrate and DRAQ5 were purchased from Thermo Fisher Scientific (Shanghai, China).

Wang Y., Zhang Y., Zhang C., Hu M., Yan Q., Zhao H., Zhang X, & Wu Y. (2020). Cholesterol-Rich Lipid Rafts in the Cellular Membrane Play an Essential Role in Avian Reovirus Replication. Frontiers in Microbiology, 11, 597794.

+ Open protocol

+ Expand

Synthesis and Characterization of Polymer Nanoparticles

Check if the same lab product or an alternative is used in the 5 most similar protocols

The
NIPAAm provided by
KJ Chemicals Co. (Tokyo, Japan) was purified by recrystallization
from n-hexane and dried at room 25 °C in vacuo.
The N,N′-dimethylaminopropylacrylamide
(DMAPAAm) was purchased from KJ Chemicals Co. (Tokyo, Japan), and
2,2′-azobisisobutyronitrile (AIBN), 3-mercaptopropionic acid
(MPA), sucrose, cytochalasin D, nocodazole, and DOPE were purchased
from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). N-Hydroxysuccinimide (NHS) and N,N′-dicyclohexylcarbodiimide (DCC) were purchased from Kanto
Chemical Co. (Tokyo, Japan). 5(6)-CF and chlorpromazine were purchased
from Sigma-Aldrich Corp. (St. Louis, MO). Filipin III was purchased
from Cayman Chemical (Ann Arbor, MI). Dulbecco’s modified Eagle’s
medium was purchased from Thermo Fisher Scientific (Waltham, CA),
and the fetal bovine serum (FBS) was purchased from Biosera (Sussex,
U.K.). The rhodamine B 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine
triethylammonium salt (rhodamine–DHPE) was purchased from Life
Technologies Co. (Carlsbad, CA), and DOTAP and N-[methoxy
(PEG) 2000]–distearoylphosphatidylethanolamine (PEG–DSPE)
were purchased from Avanti Polar Lipids Inc. (Alabaster, AL). All
other reagents and solvents were of analytical grade.

Wang J., Ayano E., Maitani Y, & Kanazawa H. (2017). Tunable Surface Properties of Temperature-Responsive Polymer-Modified Liposomes Induce Faster Cellular Uptake. ACS Omega, 2(1), 316-325.

+ Open protocol

+ Expand

Visualizing Free Cholesterol in Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

The distribution of free cholesterol within cells was visualized by staining cells with filipin‐III, a fluorescent polyene antibiotic that binds specifically to free (unesterified) cholesterol. GBM cells grown on coverslips were treated with 5 μg·mL⁻¹ filipin‐III (Cayman, Ann Arbor, MI, USA) in PBS at RT for 30 min after fixation with 4% PFA. The stained cells were then observed under LSM700 confocal microscope (Carl Zeiss), and the images were analyzed with imagej software.

Lee S.J., Choi Y., Kim H.I., Moon H.E., Paek S.H., Kim T.Y, & Ko S. (2021). Platycodin D inhibits autophagy and increases glioblastoma cell death via LDLR upregulation. Molecular Oncology, 16(1), 250-268.

+ Open protocol

+ Expand

Cellular Cholesterol Quantification

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cellular-free cholesterol content was measured using the cholesterol cell-based detection assay kit (Cayman). Co-cultured CD4+ T cells were fixed and permeabilized using the true-nuclear transcription factor buffer set (BioLegend), as per manufacturer’s instructions. Then the cells were stained with filipin III (Cayman), PE anti-human FOXP3 (BioLegend), and APC anti-human CTLA4 (BioLegend). The median filipin III intensity in FOXP3-/CTLA4-, FOXP3 + /CTLA4- and FOXP3+/CTLA4+ cells was measured by the BD Aria SORP flow cytometer. For the oxidation-based quantification of cholesterol, total cholesterol was extracted using the cholesterol extraction kit (Sigma), and then oxidized using the Amplex Red Cholesterol Assay Kit (Invitrogen), per manufacturer’s instructions. The oxidized cholesterol was analyzed by the CLARIOstar Plus fluorescence plate reader.

Gong L., Luo J., Zhang Y., Yang Y., Li S., Fang X., Zhang B., Huang J., Chow L.K., Chung D., Huang J., Huang C., Liu Q., Bai L., Tiu Y.C., Wu P., Wang Y., Tsao G.S., Kwong D.L., Lee A.W., Dai W, & Guan X.Y. (2023). Nasopharyngeal carcinoma cells promote regulatory T cell development and suppressive activity via CD70-CD27 interaction. Nature Communications, 14, 1912.

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!