Structured Review

Cayman Chemical filipin
CPPs induce <t>filipin-positive</t> puncta in an endocytosis-dependent manner. A and B , HEK293 cells were cultured with BAPTA-AM (10 μ m ), MβCD (2.5 m m ), or dynasore (50 μ m ) for 30 min followed by CPP treatment for 4 h. Cells were then
Filipin, supplied by Cayman Chemical, used in various techniques. Bioz Stars score: 93/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Images

1) Product Images from "Autophagy Induced by Calcium Phosphate Precipitates Targets Damaged Endosomes *"

Article Title: Autophagy Induced by Calcium Phosphate Precipitates Targets Damaged Endosomes *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M113.531855

CPPs induce filipin-positive puncta in an endocytosis-dependent manner. A and B , HEK293 cells were cultured with BAPTA-AM (10 μ m ), MβCD (2.5 m m ), or dynasore (50 μ m ) for 30 min followed by CPP treatment for 4 h. Cells were then
Figure Legend Snippet: CPPs induce filipin-positive puncta in an endocytosis-dependent manner. A and B , HEK293 cells were cultured with BAPTA-AM (10 μ m ), MβCD (2.5 m m ), or dynasore (50 μ m ) for 30 min followed by CPP treatment for 4 h. Cells were then

Techniques Used: Cell Culture, Conditioned Place Preference

2) Product Images from "Cholesterol and ORP1L-mediated ER contact sites control autophagosome transport and fusion with the endocytic pathway"

Article Title: Cholesterol and ORP1L-mediated ER contact sites control autophagosome transport and fusion with the endocytic pathway

Journal: Nature Communications

doi: 10.1038/ncomms11808

Cholesterol modulates autophagosome positioning. ( a ) HeLa or MelJuSo cells were stained for LC3 and 4,6-diamidino-2-phenylindole (DAPI), or with filipin to detect cholesterol. Scale bar, 10 μm. Panels below: quantification for the presence of a detectable perinuclear LC3 cluster: > 100 cells were analysed per experiment. For filipin labelling, signal intensity was measured for at least three fields per experiment and was normalized to signal intensity in HeLa cells, which was set at 1. Bars indicate mean+s.d. from independent triplicates, significance calculated using Student's t -test. ( b ) MelJuSo cells cultured for 24 h under cholesterol-depleting conditions (see Methods) or exposed for 24 h to 3 μM U18666 were stained for LC3 and DAPI. Scale bar, 10 μm. Lower panel: quantification of the average distance (μm) of the AVs from the nucleus (using the LasAF software), from > 10 cells per experiment. Bars indicate mean+s.d. from independent triplicates, significance calculated using Student's t -test. ( c ) Distribution and dynamics of mCherry-LC3-marked vesicles in control versus lipid-depleted MelJuSo cells. Confocal image at start of time lapse is shown on the left, vesicle trajectories over a 480-s interval are displayed on the right. Colours of individual vesicle trajectories reflect maximal displacement rate (blue=0, red=0.8 μm s −1 ) achieved during the 480-s interval. Scale bar, 10 μm. ( d ) Quantification of the localization of mCherry-LC3 vesicles either or not labelling for Lysotracker in MelJuSo cells treated as indicated. At least five cells per replicate were quantified, bars indicate mean+s.d. from independent triplicates. Significance calculated using Student's t -test ( Supplementary Movies 1–3 ). NS, not significant, * P
Figure Legend Snippet: Cholesterol modulates autophagosome positioning. ( a ) HeLa or MelJuSo cells were stained for LC3 and 4,6-diamidino-2-phenylindole (DAPI), or with filipin to detect cholesterol. Scale bar, 10 μm. Panels below: quantification for the presence of a detectable perinuclear LC3 cluster: > 100 cells were analysed per experiment. For filipin labelling, signal intensity was measured for at least three fields per experiment and was normalized to signal intensity in HeLa cells, which was set at 1. Bars indicate mean+s.d. from independent triplicates, significance calculated using Student's t -test. ( b ) MelJuSo cells cultured for 24 h under cholesterol-depleting conditions (see Methods) or exposed for 24 h to 3 μM U18666 were stained for LC3 and DAPI. Scale bar, 10 μm. Lower panel: quantification of the average distance (μm) of the AVs from the nucleus (using the LasAF software), from > 10 cells per experiment. Bars indicate mean+s.d. from independent triplicates, significance calculated using Student's t -test. ( c ) Distribution and dynamics of mCherry-LC3-marked vesicles in control versus lipid-depleted MelJuSo cells. Confocal image at start of time lapse is shown on the left, vesicle trajectories over a 480-s interval are displayed on the right. Colours of individual vesicle trajectories reflect maximal displacement rate (blue=0, red=0.8 μm s −1 ) achieved during the 480-s interval. Scale bar, 10 μm. ( d ) Quantification of the localization of mCherry-LC3 vesicles either or not labelling for Lysotracker in MelJuSo cells treated as indicated. At least five cells per replicate were quantified, bars indicate mean+s.d. from independent triplicates. Significance calculated using Student's t -test ( Supplementary Movies 1–3 ). NS, not significant, * P

Techniques Used: Staining, Cell Culture, Software

3) Product Images from "Mechanism of Neuronal versus Endothelial Cell Uptake of Alzheimer's Disease Amyloid ? Protein"

Article Title: Mechanism of Neuronal versus Endothelial Cell Uptake of Alzheimer's Disease Amyloid ? Protein

Journal: PLoS ONE

doi: 10.1371/journal.pone.0004627

A–D: Uptake of fluorescein labeled Aβ40 (F-Aβ40) and Alexa Fluor® 647 labeled cholera toxin (AF647-CT), caveolae-mediated endocytosis marker, by differentiated PC12 cells following 30 min incubation. (A) F-Aβ40 uptake; (B) Uptake of AF647-CT; (C) Sparse co-localization of F-Aβ40 and AF647-CT as indicated by the white masked areas; (D) Superimposition of images A and B on the differential interference contrast (DIC) image to show the location of the fluorophores in the cells. E–G: Uptake of F-Aβ40 and AF647-CT in differentiated PC12 cells treated with filipin, which is known to inhibit caveolae mediated endocytosis. (E) Uptake of F-Aβ40; (F) Substantial reduction in AF647-CT uptake in the cells treated with filipin; (G). Superimposition of images E and F on the differential interference contrast (DIC) image to show the health of filipin treated cells.
Figure Legend Snippet: A–D: Uptake of fluorescein labeled Aβ40 (F-Aβ40) and Alexa Fluor® 647 labeled cholera toxin (AF647-CT), caveolae-mediated endocytosis marker, by differentiated PC12 cells following 30 min incubation. (A) F-Aβ40 uptake; (B) Uptake of AF647-CT; (C) Sparse co-localization of F-Aβ40 and AF647-CT as indicated by the white masked areas; (D) Superimposition of images A and B on the differential interference contrast (DIC) image to show the location of the fluorophores in the cells. E–G: Uptake of F-Aβ40 and AF647-CT in differentiated PC12 cells treated with filipin, which is known to inhibit caveolae mediated endocytosis. (E) Uptake of F-Aβ40; (F) Substantial reduction in AF647-CT uptake in the cells treated with filipin; (G). Superimposition of images E and F on the differential interference contrast (DIC) image to show the health of filipin treated cells.

Techniques Used: Labeling, Marker, Incubation

4) Product Images from "Transport of Apolipoprotein B-Containing Lipoproteins through Endothelial Cells Is Associated with Apolipoprotein E-Carrying HDL-Like Particle Formation"

Article Title: Transport of Apolipoprotein B-Containing Lipoproteins through Endothelial Cells Is Associated with Apolipoprotein E-Carrying HDL-Like Particle Formation

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms19113593

Transendothelial transport of radiolabeled apoB-LPs is inhibited by filipin and unlabeled competitor lipoproteins. ( A ): The AP side of the MAEC monolayer was incubated with 20 µg/mL (35,000 DPM) of 3 H-Ch-labeled E/B-LPs ( 3 H-Ch E/B) or 3 H-Ch-labeled LDLs ( 3 H-Ch LDL) in the presence of 800 µg/mL of unlabeled E/B-LPs (uE/B) or unlabeled LDLs (uLDLs) or in the absence of unlabeled lipoproteins as a control (Ctrl) for 24 h. ( B ): The AP side of the MAEC monolayer was pre-incubated with 20 µg/mL chlorpromazine, 5 µg/mL filipin, or 1 µL/mL dimethyl sulfoxide as a Ctrl for 1 h, followed by incubation with 20 µg/mL (167,000 DPM) of 125 I-labeled TRLs, IDLs, or LDLs for 24 h. The radioactivity in the BL medium were counted. Values represent the mean ± SE of 3–5 experiments; * p
Figure Legend Snippet: Transendothelial transport of radiolabeled apoB-LPs is inhibited by filipin and unlabeled competitor lipoproteins. ( A ): The AP side of the MAEC monolayer was incubated with 20 µg/mL (35,000 DPM) of 3 H-Ch-labeled E/B-LPs ( 3 H-Ch E/B) or 3 H-Ch-labeled LDLs ( 3 H-Ch LDL) in the presence of 800 µg/mL of unlabeled E/B-LPs (uE/B) or unlabeled LDLs (uLDLs) or in the absence of unlabeled lipoproteins as a control (Ctrl) for 24 h. ( B ): The AP side of the MAEC monolayer was pre-incubated with 20 µg/mL chlorpromazine, 5 µg/mL filipin, or 1 µL/mL dimethyl sulfoxide as a Ctrl for 1 h, followed by incubation with 20 µg/mL (167,000 DPM) of 125 I-labeled TRLs, IDLs, or LDLs for 24 h. The radioactivity in the BL medium were counted. Values represent the mean ± SE of 3–5 experiments; * p

Techniques Used: Incubation, Labeling, Radioactivity

5) Product Images from "Transport of Apolipoprotein B-Containing Lipoproteins through Endothelial Cells Is Associated with Apolipoprotein E-Carrying HDL-Like Particle Formation"

Article Title: Transport of Apolipoprotein B-Containing Lipoproteins through Endothelial Cells Is Associated with Apolipoprotein E-Carrying HDL-Like Particle Formation

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms19113593

Transendothelial transport of radiolabeled apoB-LPs is inhibited by filipin and unlabeled competitor lipoproteins. ( A ): The AP side of the MAEC monolayer was incubated with 20 µg/mL (35,000 DPM) of 3 H-Ch-labeled E/B-LPs ( 3 H-Ch E/B) or 3 H-Ch-labeled LDLs ( 3 H-Ch LDL) in the presence of 800 µg/mL of unlabeled E/B-LPs (uE/B) or unlabeled LDLs (uLDLs) or in the absence of unlabeled lipoproteins as a control (Ctrl) for 24 h. ( B ): The AP side of the MAEC monolayer was pre-incubated with 20 µg/mL chlorpromazine, 5 µg/mL filipin, or 1 µL/mL dimethyl sulfoxide as a Ctrl for 1 h, followed by incubation with 20 µg/mL (167,000 DPM) of 125 I-labeled TRLs, IDLs, or LDLs for 24 h. The radioactivity in the BL medium were counted. Values represent the mean ± SE of 3–5 experiments; * p
Figure Legend Snippet: Transendothelial transport of radiolabeled apoB-LPs is inhibited by filipin and unlabeled competitor lipoproteins. ( A ): The AP side of the MAEC monolayer was incubated with 20 µg/mL (35,000 DPM) of 3 H-Ch-labeled E/B-LPs ( 3 H-Ch E/B) or 3 H-Ch-labeled LDLs ( 3 H-Ch LDL) in the presence of 800 µg/mL of unlabeled E/B-LPs (uE/B) or unlabeled LDLs (uLDLs) or in the absence of unlabeled lipoproteins as a control (Ctrl) for 24 h. ( B ): The AP side of the MAEC monolayer was pre-incubated with 20 µg/mL chlorpromazine, 5 µg/mL filipin, or 1 µL/mL dimethyl sulfoxide as a Ctrl for 1 h, followed by incubation with 20 µg/mL (167,000 DPM) of 125 I-labeled TRLs, IDLs, or LDLs for 24 h. The radioactivity in the BL medium were counted. Values represent the mean ± SE of 3–5 experiments; * p

Techniques Used: Incubation, Labeling, Radioactivity

6) Product Images from "Activation of Autophagy of Aggregation-prone Ubiquitinated Proteins by Timosaponin A-III *"

Article Title: Activation of Autophagy of Aggregation-prone Ubiquitinated Proteins by Timosaponin A-III *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.202531

TAIII-induced autophagy and cholesterol localization. A , HeLa cells expressing tfLC3 were treated with 10 μ m TAIII or DMSO vehicle (control) for 18 h. Cells were fixed and stained with 5 μg/ml of filipin, and then examined with fluorescence
Figure Legend Snippet: TAIII-induced autophagy and cholesterol localization. A , HeLa cells expressing tfLC3 were treated with 10 μ m TAIII or DMSO vehicle (control) for 18 h. Cells were fixed and stained with 5 μg/ml of filipin, and then examined with fluorescence

Techniques Used: Expressing, Staining, Fluorescence

7) Product Images from "T Cell Signal Regulation by the Actin Cytoskeleton *"

Article Title: T Cell Signal Regulation by the Actin Cytoskeleton *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M109.097311

Cholesterol- and cytoskeleton-dependent properties of probe co-clustering in the IS. A , confocal ( left ) and DIC ( right ) images of conjugated Jurkat T cells expressing L 10 -YFP. The conjugates were untreated or treated with either filipin, Lat B, or jasplakinolide
Figure Legend Snippet: Cholesterol- and cytoskeleton-dependent properties of probe co-clustering in the IS. A , confocal ( left ) and DIC ( right ) images of conjugated Jurkat T cells expressing L 10 -YFP. The conjugates were untreated or treated with either filipin, Lat B, or jasplakinolide

Techniques Used: Expressing

Jasplakinolide maintains IS lipid condensation in the presence of filipin. A , GP images of Laurdan fluorescence in labeled Jurkat cells conjugated to SEE-pulsed B cells. The samples were untreated, treated with filipin alone, or pretreated with jasplakinolide
Figure Legend Snippet: Jasplakinolide maintains IS lipid condensation in the presence of filipin. A , GP images of Laurdan fluorescence in labeled Jurkat cells conjugated to SEE-pulsed B cells. The samples were untreated, treated with filipin alone, or pretreated with jasplakinolide

Techniques Used: Fluorescence, Labeling

Filipin-dependent deregulation of Lck in the IS is blocked by jasplakinolide. A and B , confocal ( left ) and DIC ( right ) images of T cells conjugated to SEE-pulsed B cells and treated as indicated. The conjugates were stained with antibodies specific to
Figure Legend Snippet: Filipin-dependent deregulation of Lck in the IS is blocked by jasplakinolide. A and B , confocal ( left ) and DIC ( right ) images of T cells conjugated to SEE-pulsed B cells and treated as indicated. The conjugates were stained with antibodies specific to

Techniques Used: Staining

Filipin-dependent targeting of CD45 to the IS is blocked by jasplakinolide. A , confocal ( left ) and DIC ( right ) images of CD45-labeled T cells conjugated to SEE-pulsed B cells. The conjugates received the indicated treatments before fixation. The asterisks
Figure Legend Snippet: Filipin-dependent targeting of CD45 to the IS is blocked by jasplakinolide. A , confocal ( left ) and DIC ( right ) images of CD45-labeled T cells conjugated to SEE-pulsed B cells. The conjugates received the indicated treatments before fixation. The asterisks

Techniques Used: Labeling

8) Product Images from "Generation of patient specific human neural stem cells from Niemann-Pick disease type C patient-derived fibroblasts"

Article Title: Generation of patient specific human neural stem cells from Niemann-Pick disease type C patient-derived fibroblasts

Journal: Oncotarget

doi: 10.18632/oncotarget.19976

Rescue of cholesterol accumulation and self-renewal ability by VPA treatment (A) NPC-iNSCs were treated with VPA (1mM), L-NAME (100μM), and SB202190 (1μM) for 3 days. (B) VPA was treated to NPC-iNSCs at various incubation time. The relative intensity of filipin was analyzed and normalized to WT-iNSCs. (C) VPA treatment had effect on reduction of cholesterol accumulation in U18-treated WT- and NPC-iNSCs, scale bar = 50 μm. (D) VPA-treated NPC-iNSCs were stained with NPC1 and LAMP-1, scale bar = 50 μm. The quantification in (D) was conducted following the same method as in Figure 3C . (E) Analysis of neurosphere formation after VPA treatment. Impaired self-renewal ability was rescued by VPA treatment, scale bar = 100 μm. * P
Figure Legend Snippet: Rescue of cholesterol accumulation and self-renewal ability by VPA treatment (A) NPC-iNSCs were treated with VPA (1mM), L-NAME (100μM), and SB202190 (1μM) for 3 days. (B) VPA was treated to NPC-iNSCs at various incubation time. The relative intensity of filipin was analyzed and normalized to WT-iNSCs. (C) VPA treatment had effect on reduction of cholesterol accumulation in U18-treated WT- and NPC-iNSCs, scale bar = 50 μm. (D) VPA-treated NPC-iNSCs were stained with NPC1 and LAMP-1, scale bar = 50 μm. The quantification in (D) was conducted following the same method as in Figure 3C . (E) Analysis of neurosphere formation after VPA treatment. Impaired self-renewal ability was rescued by VPA treatment, scale bar = 100 μm. * P

Techniques Used: Incubation, Staining

NPC-iNSCs display disease-specific phenotypes of NPC disease (A) Scheme of neurosphere-forming assay. (B) Phase-contrast images of the WT- and NPC-neurospheres (left), scale bar = 100 μm. Self-renewal ability is characterized by the diameter (middle) and number (right) of primary and secondary neurospheres. (C) WT- and NPC-iNSCs were stained with antibodies against NPC1 and LAMP-1. Nuclei were counterstained with DAPI,scale bar = 50 μm, zoom scale bar = 10 μm. The quantification of NPC1-postivie cells was conducted following the same method used in Figure 2C . The expression levels of LAMP-1 was measured using Image J software and the value of control is set at 1. (D) Unesterified cholesterol of WT- and NPC-iNSCs was detected by filipin staining, scale bar = 50 μm. * P
Figure Legend Snippet: NPC-iNSCs display disease-specific phenotypes of NPC disease (A) Scheme of neurosphere-forming assay. (B) Phase-contrast images of the WT- and NPC-neurospheres (left), scale bar = 100 μm. Self-renewal ability is characterized by the diameter (middle) and number (right) of primary and secondary neurospheres. (C) WT- and NPC-iNSCs were stained with antibodies against NPC1 and LAMP-1. Nuclei were counterstained with DAPI,scale bar = 50 μm, zoom scale bar = 10 μm. The quantification of NPC1-postivie cells was conducted following the same method used in Figure 2C . The expression levels of LAMP-1 was measured using Image J software and the value of control is set at 1. (D) Unesterified cholesterol of WT- and NPC-iNSCs was detected by filipin staining, scale bar = 50 μm. * P

Techniques Used: Neurosphere Assay, Staining, Expressing, Software

U18 treatment leads to impairments of self-renewal and neuronal differentiation in WT-iNSCs through abnormal cholesterol accumulation (A) Representative filipin staining results indicates that U18 treatment induced cholesterol accumulation, compared to non-treated WT-iNSC, scale bar = 50 μm. U18 was treated at various concentration to determine the appropriate concentration. The intensity of filipin was analyzed and quantified. (B) U18-treated WT-iNSCs were stained with antibody against LAMP-1 and quantified using the same method as in Figure 3C , scale bar = 50 μm, zoom scale bar = 10 μm. (C) Phase-contrast images of the control- and U18-treated WT-neurospheres, scale bar = 100 μm (left). Self-renewal ability of U18-treated WT-neurosphere was characterized by the size (middle) and number (right) of neurospheres. (D) After U18 treatment, WT-iNSCs were differentiated into neurons (α-INTERNEXIN, NF, and TUJ1). Nuclei were counterstained with DAPI, scale bar = 50 μm, zoom scale bar = 10 μm. (E) U18-treated WT-iNSCs and non-treated WT-iNSCs were differentiated into GFAP-positive astrocytes. Nuclei were counterstained with DAPI, scale bar = 50 μm. Differentiation capacity into neurons (D) and astrocytes (E) was quantified using the same method as in Figure 2C . * P
Figure Legend Snippet: U18 treatment leads to impairments of self-renewal and neuronal differentiation in WT-iNSCs through abnormal cholesterol accumulation (A) Representative filipin staining results indicates that U18 treatment induced cholesterol accumulation, compared to non-treated WT-iNSC, scale bar = 50 μm. U18 was treated at various concentration to determine the appropriate concentration. The intensity of filipin was analyzed and quantified. (B) U18-treated WT-iNSCs were stained with antibody against LAMP-1 and quantified using the same method as in Figure 3C , scale bar = 50 μm, zoom scale bar = 10 μm. (C) Phase-contrast images of the control- and U18-treated WT-neurospheres, scale bar = 100 μm (left). Self-renewal ability of U18-treated WT-neurosphere was characterized by the size (middle) and number (right) of neurospheres. (D) After U18 treatment, WT-iNSCs were differentiated into neurons (α-INTERNEXIN, NF, and TUJ1). Nuclei were counterstained with DAPI, scale bar = 50 μm, zoom scale bar = 10 μm. (E) U18-treated WT-iNSCs and non-treated WT-iNSCs were differentiated into GFAP-positive astrocytes. Nuclei were counterstained with DAPI, scale bar = 50 μm. Differentiation capacity into neurons (D) and astrocytes (E) was quantified using the same method as in Figure 2C . * P

Techniques Used: Staining, Concentration Assay

9) Product Images from "Retinal Vascular Abnormalities and Microglia Activation in Mice with Deficiency in Cytochrome P450 46A1–Mediated Cholesterol Removal"

Article Title: Retinal Vascular Abnormalities and Microglia Activation in Mice with Deficiency in Cytochrome P450 46A1–Mediated Cholesterol Removal

Journal: The American Journal of Pathology

doi: 10.1016/j.ajpath.2018.10.013

Retinal detection of unesterified and esterified cholesterol with filipin. Representative images are shown. A and E: Stains (cyan) for unesterified cholesterol (these sections were treated with filipin). B and F: Control stains for completeness of removal of unesterified cholesterol (these sections were extracted with 70% aqueous ethanol and treated then with filipin). C and G: Control stains for background fluorescence (these sections were extracted with ethanol but not treated with filipin). D and H: Stains (cyan) for esterified cholesterol (these sections were extracted with 70% ethanol and then sequentially treated with cholesterol esterase and filipin). B–D and F–H: Panels for esterified cholesterol consist of phase contrast images ( left panels ) and histochemistry images ( right panels ). n = 3 ( A–H ). Scale bars = 50 μm ( A–H ). GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; IS, photoreceptor inner segment; ONL, outer nuclear layer; OPL, outer plexiform layer; OS, photoreceptor outer segment; RPE, retinal pigment epithelium.
Figure Legend Snippet: Retinal detection of unesterified and esterified cholesterol with filipin. Representative images are shown. A and E: Stains (cyan) for unesterified cholesterol (these sections were treated with filipin). B and F: Control stains for completeness of removal of unesterified cholesterol (these sections were extracted with 70% aqueous ethanol and treated then with filipin). C and G: Control stains for background fluorescence (these sections were extracted with ethanol but not treated with filipin). D and H: Stains (cyan) for esterified cholesterol (these sections were extracted with 70% ethanol and then sequentially treated with cholesterol esterase and filipin). B–D and F–H: Panels for esterified cholesterol consist of phase contrast images ( left panels ) and histochemistry images ( right panels ). n = 3 ( A–H ). Scale bars = 50 μm ( A–H ). GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; IS, photoreceptor inner segment; ONL, outer nuclear layer; OPL, outer plexiform layer; OS, photoreceptor outer segment; RPE, retinal pigment epithelium.

Techniques Used: Fluorescence

10) Product Images from "Caveolin-1 mediates endotoxin inhibition of endothelin-1-induced endothelial nitric oxide synthase activity in liver sinusoidal endothelial cells"

Article Title: Caveolin-1 mediates endotoxin inhibition of endothelin-1-induced endothelial nitric oxide synthase activity in liver sinusoidal endothelial cells

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

doi: 10.1152/ajpgi.00106.2009

eNOS activity after filipin treatments. Following treatment of LSECs with vehicle, 0.05, 0.1, 0.2, 0.25, 0.5, 1, and 5 μg/ml filipin for 30 min, basal eNOS activity ( A ) was measured. * P
Figure Legend Snippet: eNOS activity after filipin treatments. Following treatment of LSECs with vehicle, 0.05, 0.1, 0.2, 0.25, 0.5, 1, and 5 μg/ml filipin for 30 min, basal eNOS activity ( A ) was measured. * P

Techniques Used: Activity Assay

11) Product Images from "Cytoskeletal Modulation of Lipid Interactions Regulates Lck Kinase Activity *"

Article Title: Cytoskeletal Modulation of Lipid Interactions Regulates Lck Kinase Activity *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M111.320747

Neomycin with filipin disrupts co-clustering of L o and L d phase probes. Shown is the FRET efficiency ( A ) and K values ( B ) determined for untreated control cells, cells treated with neomycin ( Neo ) alone, and cells treated with filipin following pretreatment
Figure Legend Snippet: Neomycin with filipin disrupts co-clustering of L o and L d phase probes. Shown is the FRET efficiency ( A ) and K values ( B ) determined for untreated control cells, cells treated with neomycin ( Neo ) alone, and cells treated with filipin following pretreatment

Techniques Used:

Neomycin, neomycin co-treatment with filipin, and blebbistatin do not affect the F-actin content in Jurkat cells. A , cells were stained with Texas Red-labeled phalloidin (Phalloidin-Texas Red) following the respective treatments, and the labeling was
Figure Legend Snippet: Neomycin, neomycin co-treatment with filipin, and blebbistatin do not affect the F-actin content in Jurkat cells. A , cells were stained with Texas Red-labeled phalloidin (Phalloidin-Texas Red) following the respective treatments, and the labeling was

Techniques Used: Staining, Labeling

12) Product Images from "Functional inhibition of acid sphingomyelinase disrupts infection by intracellular bacterial pathogens"

Article Title: Functional inhibition of acid sphingomyelinase disrupts infection by intracellular bacterial pathogens

Journal: Life Science Alliance

doi: 10.26508/lsa.201800292

C. trachomatis and C. pneumoniae exhibit reduced FIASMA sensitivity. (A–D) Desipramine or DMSO-treated HeLa cells infected with C. trachomatis (Ctr; A, B) or C. pneumoniae (Cpn; C, D) were either lysed to recover infectious progeny that were incubated with naïve HeLa cells to determine inclusion-forming units (A, C) or were fixed and assessed by immunofluorescence microscopy to determine inclusion area (B, D). (E–J) Desipramine-treated or control cells were infected with Ctr (E, G, I) or Cpn (F, H, J). The cells were either incubated with filipin (E, F) or screened with antibodies specific for LBPA (G, H) or CERT (I, J) together with antisera against C. trachomatis (E, G, I) or C. pneumoniae (F, H, J). DAPI or DRAQ5 was used to stain DNA. Regions that are demarcated by hatched-line boxes are magnified in the inset panels. Error bars indicate SD. t test was used to test for a significant difference among pairs. Statistically significant ( *P
Figure Legend Snippet: C. trachomatis and C. pneumoniae exhibit reduced FIASMA sensitivity. (A–D) Desipramine or DMSO-treated HeLa cells infected with C. trachomatis (Ctr; A, B) or C. pneumoniae (Cpn; C, D) were either lysed to recover infectious progeny that were incubated with naïve HeLa cells to determine inclusion-forming units (A, C) or were fixed and assessed by immunofluorescence microscopy to determine inclusion area (B, D). (E–J) Desipramine-treated or control cells were infected with Ctr (E, G, I) or Cpn (F, H, J). The cells were either incubated with filipin (E, F) or screened with antibodies specific for LBPA (G, H) or CERT (I, J) together with antisera against C. trachomatis (E, G, I) or C. pneumoniae (F, H, J). DAPI or DRAQ5 was used to stain DNA. Regions that are demarcated by hatched-line boxes are magnified in the inset panels. Error bars indicate SD. t test was used to test for a significant difference among pairs. Statistically significant ( *P

Techniques Used: Infection, Incubation, Immunofluorescence, Microscopy, Staining

Desipramine-induced cholesterol accumulation in the C. burnetii vacuole is bactericidal. (A, B) mCherry- C. burnetii (Cb)–infected THP-1 macrophage-like cells (A) or mCherry- C. burnetii grown in axenic medium (B) were treated with desipramine or not treated with. The bacterial load was measured as relative fluorescent units. (C–E) C. burnetii was added to HeLa cells (C, E) or MH-S cells (D) that had been pretreated with desipramine or DMSO, or C. burnetii –infected cells were treated at the indicated days postinfection (E). Bacterial load was measured using a CFU assay. (F) CCV area was determined for desipramine and DMSO-treated C. burnetii –infected HeLa cells. (G) HeLa cells that had been treated with desipramine and infected with mCherry- C. burnetii were labeled with filipin and CD63 antibody. Error bars indicate SD. t test was used to test for a significant difference among pairs. Statistically significant ( **P
Figure Legend Snippet: Desipramine-induced cholesterol accumulation in the C. burnetii vacuole is bactericidal. (A, B) mCherry- C. burnetii (Cb)–infected THP-1 macrophage-like cells (A) or mCherry- C. burnetii grown in axenic medium (B) were treated with desipramine or not treated with. The bacterial load was measured as relative fluorescent units. (C–E) C. burnetii was added to HeLa cells (C, E) or MH-S cells (D) that had been pretreated with desipramine or DMSO, or C. burnetii –infected cells were treated at the indicated days postinfection (E). Bacterial load was measured using a CFU assay. (F) CCV area was determined for desipramine and DMSO-treated C. burnetii –infected HeLa cells. (G) HeLa cells that had been treated with desipramine and infected with mCherry- C. burnetii were labeled with filipin and CD63 antibody. Error bars indicate SD. t test was used to test for a significant difference among pairs. Statistically significant ( **P

Techniques Used: Infection, Colony-forming Unit Assay, Labeling

13) Product Images from "Small molecule inhibitors reveal Niemann-Pick C1 is essential for ebolavirus infection"

Article Title: Small molecule inhibitors reveal Niemann-Pick C1 is essential for ebolavirus infection

Journal: Nature

doi: 10.1038/nature10380

NPC1 is essential for ebolavirus infection a , HeLa cells were treated with 3.0 (20 μM), 3.47 (1.25 μM) or vehicle for 18 hours, then fixed and incubated with the cholesterol-avid fluorophore filipin. b , HeLa cells were transfected with siRNAs targeting ASM, Alix, NPC1, NPC2, and ORP5. After 72 hours, VSV EboV GP or LFV GP infection of these cells was measured as in Fig 1c . Data is mean ± s.d. (n=3) and is representative of 3 experiments. c , CHO wt , CHO null and CHO null cells stably expressing mouse NPC1 (CHO NPC1 ) or NPC1 mutants L657F, P692S, D787N were exposed to MLV particles encoding LacZ and pseudotyped with either EboV GP or VSV G. Results are the mean ± s.d. (n=4) and is representative of 3 experiments. d , CHO wt , CHO null , and CHO NPC1 cells were infected with replication competent ebolavirus Zaire-Mayinga encoding GFP (moi = 1). Results are mean relative fluorescence units ± s.d. (n=3). e , CHO wt and CHO null cells were treated with the cathepsin B inhibitor CA074 (80 μM) or vehicle. These cells were challenged with VSV G particles or VSV EboV GP particles treated with thermolysin (EboV GP THL ) or untreated control (EboV GP). Infection was measured as in Fig 1b . Data is mean ± s.d. (n=9).
Figure Legend Snippet: NPC1 is essential for ebolavirus infection a , HeLa cells were treated with 3.0 (20 μM), 3.47 (1.25 μM) or vehicle for 18 hours, then fixed and incubated with the cholesterol-avid fluorophore filipin. b , HeLa cells were transfected with siRNAs targeting ASM, Alix, NPC1, NPC2, and ORP5. After 72 hours, VSV EboV GP or LFV GP infection of these cells was measured as in Fig 1c . Data is mean ± s.d. (n=3) and is representative of 3 experiments. c , CHO wt , CHO null and CHO null cells stably expressing mouse NPC1 (CHO NPC1 ) or NPC1 mutants L657F, P692S, D787N were exposed to MLV particles encoding LacZ and pseudotyped with either EboV GP or VSV G. Results are the mean ± s.d. (n=4) and is representative of 3 experiments. d , CHO wt , CHO null , and CHO NPC1 cells were infected with replication competent ebolavirus Zaire-Mayinga encoding GFP (moi = 1). Results are mean relative fluorescence units ± s.d. (n=3). e , CHO wt and CHO null cells were treated with the cathepsin B inhibitor CA074 (80 μM) or vehicle. These cells were challenged with VSV G particles or VSV EboV GP particles treated with thermolysin (EboV GP THL ) or untreated control (EboV GP). Infection was measured as in Fig 1b . Data is mean ± s.d. (n=9).

Techniques Used: Infection, Incubation, Transfection, Stable Transfection, Expressing, Fluorescence

14) Product Images from "2-Hydroxypropyl-beta-cyclodextrin (HPβCD) reduces age-related lipofuscin accumulation through a cholesterol-associated pathway"

Article Title: 2-Hydroxypropyl-beta-cyclodextrin (HPβCD) reduces age-related lipofuscin accumulation through a cholesterol-associated pathway

Journal: Scientific Reports

doi: 10.1038/s41598-017-02387-8

Long term HPβCD treatment increases cellular cholesterol. ( Top Row ) - Filipin staining shows HPβCD treatment, LF loading and the two combined each cause substantial cellular cholesterol increase relative to healthy cells. ( Bottom Row ) Cells with increased lysosome cholesterol composition are more resistant to apoptosis induced by the lysomotropic agent MSDH. Healthy confluent cells were killed by MSDH exposure (45 uM MSDH for 42 hours) while HPβCD treated cells and LF loaded cells survived with no signs of visual stress. Therefore, HPβCD and LF modulate lysosome membrane cholesterol composition in a manner consistent with that suggested by filipin staining.
Figure Legend Snippet: Long term HPβCD treatment increases cellular cholesterol. ( Top Row ) - Filipin staining shows HPβCD treatment, LF loading and the two combined each cause substantial cellular cholesterol increase relative to healthy cells. ( Bottom Row ) Cells with increased lysosome cholesterol composition are more resistant to apoptosis induced by the lysomotropic agent MSDH. Healthy confluent cells were killed by MSDH exposure (45 uM MSDH for 42 hours) while HPβCD treated cells and LF loaded cells survived with no signs of visual stress. Therefore, HPβCD and LF modulate lysosome membrane cholesterol composition in a manner consistent with that suggested by filipin staining.

Techniques Used: Staining

15) Product Images from "Elevated Cholesterol in the Coxiella burnetii Intracellular Niche Is Bacteriolytic"

Article Title: Elevated Cholesterol in the Coxiella burnetii Intracellular Niche Is Bacteriolytic

Journal: mBio

doi: 10.1128/mBio.02313-16

Altered cellular cholesterol homeostasis is bactericidal. (A) Microscopy images showing that U18666A treatment traps cholesterol in the C. burnetii PV in HeLa cells. mCherry- C. burnetii -infected HeLa cells were treated with 5 µM U18666A for 6 h, fixed, and stained for sterols (filipin) and PV (LAMP-1). Compared to mock-treated cells, there is an increase in filipin labeling in and around the PV following treatment with U18666A. The white arrows point to the PVs. Filipin intensity is shown as a heat map, with yellow showing the highest filipin intensity and blue showing the lowest filipin intensity. Bars = 5 µm. (B) Quantitation of lytic PVs in U18666A-treated cells after treatment of mCherry- C. burnetii- infected HeLa cells with 1 or 5 µM U18666A. PVs were scored for the presence (lytic) or absence (nonlytic) of free mCherry in the PV lumen, resulting from the lysis of mCherry-expressing bacteria. The means plus SEM (error bars) from three individual experiments are shown. The means were compared to the value with no cholesterol by one-way ANOVA with Dunnett’s posthoc test, and statistically different values are indicated by asterisks as follows: *, P
Figure Legend Snippet: Altered cellular cholesterol homeostasis is bactericidal. (A) Microscopy images showing that U18666A treatment traps cholesterol in the C. burnetii PV in HeLa cells. mCherry- C. burnetii -infected HeLa cells were treated with 5 µM U18666A for 6 h, fixed, and stained for sterols (filipin) and PV (LAMP-1). Compared to mock-treated cells, there is an increase in filipin labeling in and around the PV following treatment with U18666A. The white arrows point to the PVs. Filipin intensity is shown as a heat map, with yellow showing the highest filipin intensity and blue showing the lowest filipin intensity. Bars = 5 µm. (B) Quantitation of lytic PVs in U18666A-treated cells after treatment of mCherry- C. burnetii- infected HeLa cells with 1 or 5 µM U18666A. PVs were scored for the presence (lytic) or absence (nonlytic) of free mCherry in the PV lumen, resulting from the lysis of mCherry-expressing bacteria. The means plus SEM (error bars) from three individual experiments are shown. The means were compared to the value with no cholesterol by one-way ANOVA with Dunnett’s posthoc test, and statistically different values are indicated by asterisks as follows: *, P

Techniques Used: Microscopy, Infection, Staining, Labeling, Quantitation Assay, Lysis, Expressing

Related Articles

Microscopy:

Article Title: Autophagy Induced by Calcium Phosphate Precipitates Targets Damaged Endosomes *
Article Snippet: .. Alternatively, fixed cells were incubated with filipin (0.1 mg/ml; Cayman Chemicals, Ann Arbor, MI) without permeabilization for 4 h. Fluorescence microscopy was carried out with a Nikon Eclipse TE200 microscope with the NIS-Elements software. .. Confocal microscopy was carried out with PerkinElmer Life Sciences R2-E2 equipped with Andor iQ software for colocalization studies.

Article Title: Cholesterol and ORP1L-mediated ER contact sites control autophagosome transport and fusion with the endocytic pathway
Article Snippet: Twenty-four hours later, cells were fixed in 3.7% formaldehyde for 10 min and permeabilized in ice-cold methanol for 2 min. Staining was performed with the antibodies mentioned above and 4,6-diamidino-2-phenylindole (Invitrogen) to stain DNA or Filipin (Cayman Chemicals) to detect cholesterol. .. Images were acquired using a Leica TCS SP5 confocal microscope (Leica Microsystems, Wetzlar, Germany) at × 63 magnification.

Transfection:

Article Title: Cholesterol and ORP1L-mediated ER contact sites control autophagosome transport and fusion with the endocytic pathway
Article Snippet: Confocal microscopy Cells were seeded on coverslips, transfected 18 h later and treated as indicated. .. Twenty-four hours later, cells were fixed in 3.7% formaldehyde for 10 min and permeabilized in ice-cold methanol for 2 min. Staining was performed with the antibodies mentioned above and 4,6-diamidino-2-phenylindole (Invitrogen) to stain DNA or Filipin (Cayman Chemicals) to detect cholesterol.

Confocal Microscopy:

Article Title: Autophagy Induced by Calcium Phosphate Precipitates Targets Damaged Endosomes *
Article Snippet: Alternatively, fixed cells were incubated with filipin (0.1 mg/ml; Cayman Chemicals, Ann Arbor, MI) without permeabilization for 4 h. Fluorescence microscopy was carried out with a Nikon Eclipse TE200 microscope with the NIS-Elements software. .. Confocal microscopy was carried out with PerkinElmer Life Sciences R2-E2 equipped with Andor iQ software for colocalization studies.

Article Title: Cholesterol and ORP1L-mediated ER contact sites control autophagosome transport and fusion with the endocytic pathway
Article Snippet: Paragraph title: Confocal microscopy ... Twenty-four hours later, cells were fixed in 3.7% formaldehyde for 10 min and permeabilized in ice-cold methanol for 2 min. Staining was performed with the antibodies mentioned above and 4,6-diamidino-2-phenylindole (Invitrogen) to stain DNA or Filipin (Cayman Chemicals) to detect cholesterol.

Fluorescence:

Article Title: Autophagy Induced by Calcium Phosphate Precipitates Targets Damaged Endosomes *
Article Snippet: .. Alternatively, fixed cells were incubated with filipin (0.1 mg/ml; Cayman Chemicals, Ann Arbor, MI) without permeabilization for 4 h. Fluorescence microscopy was carried out with a Nikon Eclipse TE200 microscope with the NIS-Elements software. .. Confocal microscopy was carried out with PerkinElmer Life Sciences R2-E2 equipped with Andor iQ software for colocalization studies.

Article Title: Spatial Distribution of the Pathways of Cholesterol Homeostasis in Human Retina
Article Snippet: Sections were removed from the freezer, air-dried for 1 hr, and rehydrated with PBS three times for 5 min. To detect unesterified cholesterol (UC), filipin III (Cayman Chemical, Ann Arbor, MI), 50 µg/ml in PBS prepared from a 3.3 mg/ml stock in dimethylsulfoxide, was applied to slides for 1 hr in a light-blocking box. .. Filipin fluorescence was excited at 340–380 nm and emission collected at 385–470 nm.

Isolation:

Article Title: Preferential uptake of antioxidant carbon nanoparticles by T lymphocytes for immunomodulation
Article Snippet: Cellular uptake of PEG-HCCs Freshly isolated rat splenocytes were resuspended (1 × 106 cells/mL) in medium + 5% FBS and incubated with or without PEG-HCCs (0.1 μg/mL) for the indicated times at 37 °C, 5% CO2 unless indicated in the figure caption. .. 25 mM sodium azide (Sigma-Aldrich) was used to inhibit active transport processes; 1 μg/mL filipin III (Cayman Chemical) was used to block caveolin-mediated endocytosis; 10 μg/mL chlorpromazine (Sigma-Aldrich) was used to inhibit clathrin-mediated endocytosis .

Cell Culture:

Article Title: Autophagy Induced by Calcium Phosphate Precipitates Targets Damaged Endosomes *
Article Snippet: Cells were cultured on glass slides, fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, and blocked with 2% BSA before incubation with the indicated antibodies. .. Alternatively, fixed cells were incubated with filipin (0.1 mg/ml; Cayman Chemicals, Ann Arbor, MI) without permeabilization for 4 h. Fluorescence microscopy was carried out with a Nikon Eclipse TE200 microscope with the NIS-Elements software.

Infection:

Article Title: Internalization and Trafficking of Nontypeable Haemophilus influenzae in Human Respiratory Epithelial Cells and Roles of IgA1 Proteases for Optimal Invasion and Persistence
Article Snippet: Noncytotoxic concentrations of each inhibitor were established through lactate dehydrogenase (LDH) release assays of uninfected cells using a cytotoxicity detection kit (Roche) and through trypan blue exclusion assays of infected cells. .. A 500 μg · ml−1 working stock of filipin III (Cayman Chemical) in DMSO was made fresh for same-day use.

Purification:

Article Title: Preferential uptake of antioxidant carbon nanoparticles by T lymphocytes for immunomodulation
Article Snippet: Cells were then washed with FCM buffer and incubated with purified mouse IgG (Life Technologies) to block FcγII/FcγIII receptors. .. 25 mM sodium azide (Sigma-Aldrich) was used to inhibit active transport processes; 1 μg/mL filipin III (Cayman Chemical) was used to block caveolin-mediated endocytosis; 10 μg/mL chlorpromazine (Sigma-Aldrich) was used to inhibit clathrin-mediated endocytosis .

Labeling:

Article Title: Mechanism of Neuronal versus Endothelial Cell Uptake of Alzheimer's Disease Amyloid ? Protein
Article Snippet: Subsequently, they were incubated in DMEM containing 15 µg/ml F-Aβ40 and 5 µg/ml Alexa Flour 647 labeled Cholera toxin (AF647-CT) (Invitrogen-Molecular Probes, Carlsbad, CA), a specific marker of caveolae internalization, for 30 min at 37°C. .. Alternatively, PC12 cells were pre-incubated for 30 min in DMEM containing filipin (5 µg/ml) (Cayman Chemical Company, Ann Arbor, MI), a sterol-binding agent that selectively inhibits caveolae invagination without affecting the function of clathrin-coated pits , followed by incubation in DMEM containing 15 µg/ml F-Aβ40 and 5 µg/ml AF647-CT for 30 min at 37°C.

Concentration Assay:

Article Title: The glycosphingolipid MacCer promotes synaptic bouton formation in Drosophila by interacting with Wnt
Article Snippet: D,L-threo-PDMP (Matreya) and filipin III (Cayman) were dissolved in DMSO and then individually added to standard media at specific concentrations. .. All the treatments used DMSO vehicle at a concentration of 0.5%.

Incubation:

Article Title: Autophagy Induced by Calcium Phosphate Precipitates Targets Damaged Endosomes *
Article Snippet: .. Alternatively, fixed cells were incubated with filipin (0.1 mg/ml; Cayman Chemicals, Ann Arbor, MI) without permeabilization for 4 h. Fluorescence microscopy was carried out with a Nikon Eclipse TE200 microscope with the NIS-Elements software. .. Confocal microscopy was carried out with PerkinElmer Life Sciences R2-E2 equipped with Andor iQ software for colocalization studies.

Article Title: Mechanism of Neuronal versus Endothelial Cell Uptake of Alzheimer's Disease Amyloid ? Protein
Article Snippet: .. Alternatively, PC12 cells were pre-incubated for 30 min in DMEM containing filipin (5 µg/ml) (Cayman Chemical Company, Ann Arbor, MI), a sterol-binding agent that selectively inhibits caveolae invagination without affecting the function of clathrin-coated pits , followed by incubation in DMEM containing 15 µg/ml F-Aβ40 and 5 µg/ml AF647-CT for 30 min at 37°C. ..

Article Title: Preferential uptake of antioxidant carbon nanoparticles by T lymphocytes for immunomodulation
Article Snippet: Cells were then washed with FCM buffer and incubated with purified mouse IgG (Life Technologies) to block FcγII/FcγIII receptors. .. 25 mM sodium azide (Sigma-Aldrich) was used to inhibit active transport processes; 1 μg/mL filipin III (Cayman Chemical) was used to block caveolin-mediated endocytosis; 10 μg/mL chlorpromazine (Sigma-Aldrich) was used to inhibit clathrin-mediated endocytosis .

other:

Article Title: Transport of Apolipoprotein B-Containing Lipoproteins through Endothelial Cells Is Associated with Apolipoprotein E-Carrying HDL-Like Particle Formation
Article Snippet: Filipin (#70440) and fetal bovine serum (FBS) were purchased from Cayman Chemical (Ann Arbor, MI, USA) and Life Technologies (Carlsbad, CA, USA), respectively.

Article Title: Uptake and function of membrane‐destabilizing cationic nanogels for intracellular drug delivery. Uptake and function of membrane‐destabilizing cationic nanogels for intracellular drug delivery
Article Snippet: Filipin III was purchased from Cayman Chemical (Ann Arbor, MI).

Article Title: Activation of Autophagy of Aggregation-prone Ubiquitinated Proteins by Timosaponin A-III *
Article Snippet: Filipin was from Cayman Chemical. p62 antibody was from BD Bioscience.

Article Title: Cell geometry dependent changes in plasma membrane order direct stem cell signalling and fate
Article Snippet: For inhibitor studies, the following concentrations were used: 2.5 µM Y27632, 40 nM Cytochalasin D (both from Sigma), 3 µM MK-2206 (Active Biochemicals), 0.5 mM methyl-β-cyclodextrin (Santa Cruz Biotechnology), 0.5 µM filipin III and 20 µM (both from Cayman Chemicals).

Blocking Assay:

Article Title: Preferential uptake of antioxidant carbon nanoparticles by T lymphocytes for immunomodulation
Article Snippet: .. 25 mM sodium azide (Sigma-Aldrich) was used to inhibit active transport processes; 1 μg/mL filipin III (Cayman Chemical) was used to block caveolin-mediated endocytosis; 10 μg/mL chlorpromazine (Sigma-Aldrich) was used to inhibit clathrin-mediated endocytosis . ..

Mass Spectrometry:

Article Title: Spatial Distribution of the Pathways of Cholesterol Homeostasis in Human Retina
Article Snippet: Previous studies also validated the use of filipin for histochemistry staining by parallel results with enzymatic, chromatographic and mass spectrometry assays (reviewed in ). .. Sections were removed from the freezer, air-dried for 1 hr, and rehydrated with PBS three times for 5 min. To detect unesterified cholesterol (UC), filipin III (Cayman Chemical, Ann Arbor, MI), 50 µg/ml in PBS prepared from a 3.3 mg/ml stock in dimethylsulfoxide, was applied to slides for 1 hr in a light-blocking box.

Marker:

Article Title: Mechanism of Neuronal versus Endothelial Cell Uptake of Alzheimer's Disease Amyloid ? Protein
Article Snippet: Subsequently, they were incubated in DMEM containing 15 µg/ml F-Aβ40 and 5 µg/ml Alexa Flour 647 labeled Cholera toxin (AF647-CT) (Invitrogen-Molecular Probes, Carlsbad, CA), a specific marker of caveolae internalization, for 30 min at 37°C. .. Alternatively, PC12 cells were pre-incubated for 30 min in DMEM containing filipin (5 µg/ml) (Cayman Chemical Company, Ann Arbor, MI), a sterol-binding agent that selectively inhibits caveolae invagination without affecting the function of clathrin-coated pits , followed by incubation in DMEM containing 15 µg/ml F-Aβ40 and 5 µg/ml AF647-CT for 30 min at 37°C.

Staining:

Article Title: Autophagy Induced by Calcium Phosphate Precipitates Targets Damaged Endosomes *
Article Snippet: Paragraph title: Immunofluorescence Staining and Fluorescence Microscopy ... Alternatively, fixed cells were incubated with filipin (0.1 mg/ml; Cayman Chemicals, Ann Arbor, MI) without permeabilization for 4 h. Fluorescence microscopy was carried out with a Nikon Eclipse TE200 microscope with the NIS-Elements software.

Article Title: Spatial Distribution of the Pathways of Cholesterol Homeostasis in Human Retina
Article Snippet: Paragraph title: Filipin staining ... Sections were removed from the freezer, air-dried for 1 hr, and rehydrated with PBS three times for 5 min. To detect unesterified cholesterol (UC), filipin III (Cayman Chemical, Ann Arbor, MI), 50 µg/ml in PBS prepared from a 3.3 mg/ml stock in dimethylsulfoxide, was applied to slides for 1 hr in a light-blocking box.

Article Title: Cholesterol and ORP1L-mediated ER contact sites control autophagosome transport and fusion with the endocytic pathway
Article Snippet: .. Twenty-four hours later, cells were fixed in 3.7% formaldehyde for 10 min and permeabilized in ice-cold methanol for 2 min. Staining was performed with the antibodies mentioned above and 4,6-diamidino-2-phenylindole (Invitrogen) to stain DNA or Filipin (Cayman Chemicals) to detect cholesterol. .. Images were acquired using a Leica TCS SP5 confocal microscope (Leica Microsystems, Wetzlar, Germany) at × 63 magnification.

Article Title: Preferential uptake of antioxidant carbon nanoparticles by T lymphocytes for immunomodulation
Article Snippet: Cells were either left intact or permeabilized with freshly prepared 0.5% saponin (EMD Millipore) in FCM buffer, and then stained for PEG-HCCs with an anti-PEG antibody and analyzed by FCM ( ). .. 25 mM sodium azide (Sigma-Aldrich) was used to inhibit active transport processes; 1 μg/mL filipin III (Cayman Chemical) was used to block caveolin-mediated endocytosis; 10 μg/mL chlorpromazine (Sigma-Aldrich) was used to inhibit clathrin-mediated endocytosis .

Immunofluorescence:

Article Title: Autophagy Induced by Calcium Phosphate Precipitates Targets Damaged Endosomes *
Article Snippet: Paragraph title: Immunofluorescence Staining and Fluorescence Microscopy ... Alternatively, fixed cells were incubated with filipin (0.1 mg/ml; Cayman Chemicals, Ann Arbor, MI) without permeabilization for 4 h. Fluorescence microscopy was carried out with a Nikon Eclipse TE200 microscope with the NIS-Elements software.

Plasmid Preparation:

Article Title: Spatial Distribution of the Pathways of Cholesterol Homeostasis in Human Retina
Article Snippet: Sections were removed from the freezer, air-dried for 1 hr, and rehydrated with PBS three times for 5 min. To detect unesterified cholesterol (UC), filipin III (Cayman Chemical, Ann Arbor, MI), 50 µg/ml in PBS prepared from a 3.3 mg/ml stock in dimethylsulfoxide, was applied to slides for 1 hr in a light-blocking box. .. Sections were removed from the freezer, air-dried for 1 hr, and rehydrated with PBS three times for 5 min. To detect unesterified cholesterol (UC), filipin III (Cayman Chemical, Ann Arbor, MI), 50 µg/ml in PBS prepared from a 3.3 mg/ml stock in dimethylsulfoxide, was applied to slides for 1 hr in a light-blocking box.

Software:

Article Title: Autophagy Induced by Calcium Phosphate Precipitates Targets Damaged Endosomes *
Article Snippet: .. Alternatively, fixed cells were incubated with filipin (0.1 mg/ml; Cayman Chemicals, Ann Arbor, MI) without permeabilization for 4 h. Fluorescence microscopy was carried out with a Nikon Eclipse TE200 microscope with the NIS-Elements software. .. Confocal microscopy was carried out with PerkinElmer Life Sciences R2-E2 equipped with Andor iQ software for colocalization studies.

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  • 93
    Cayman Chemical filipin iii
    Frequency distributions of intracellular staining of PDETB30‐OG488 in Caco‐2 cells. Cellular internalization examined in the presence of no inhibitor (a), chlorpromazine (b), <t>filipin</t> <t>III</t> (c), nystatin (d), wortmannin (e), amiloride (f), or 4 °C (g). Untreated (no PDETB30‐OG488) is shown in panel (h). Caco‐2 cells were preincubated with inhibitors for 30 min, exposed to 25 μg ml −1 PDETB30‐OG488 for 60 min, and imaged via ImageStream cytometry after 60 min further incubation. Histograms generated from image analysis of at least 500 cells
    Filipin Iii, supplied by Cayman Chemical, used in various techniques. Bioz Stars score: 93/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    filipin iii - by Bioz Stars, 2020-04
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    86
    Cayman Chemical filipin quantitative staining
    Syn acts on the content of plasma membrane cholesterol that affects calcium channel activation and neurotransmitter release. A , Bar chart showing cholesterol levels determined by quantitative fluorescent <t>filipin</t> staining. Cortical neurons incubated with
    Filipin Quantitative Staining, supplied by Cayman Chemical, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Cayman Chemical o methyl serine dodecylamide hydrochloride msdh filipin staining
    Long term HPβCD treatment increases cellular cholesterol. ( Top Row ) - <t>Filipin</t> staining shows HPβCD treatment, LF loading and the two combined each cause substantial cellular cholesterol increase relative to healthy cells. ( Bottom Row ) Cells with increased lysosome cholesterol composition are more resistant to apoptosis induced by the lysomotropic agent <t>MSDH.</t> Healthy confluent cells were killed by MSDH exposure (45 uM MSDH for 42 hours) while HPβCD treated cells and LF loaded cells survived with no signs of visual stress. Therefore, HPβCD and LF modulate lysosome membrane cholesterol composition in a manner consistent with that suggested by filipin staining.
    O Methyl Serine Dodecylamide Hydrochloride Msdh Filipin Staining, supplied by Cayman Chemical, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/o methyl serine dodecylamide hydrochloride msdh filipin staining/product/Cayman Chemical
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    Image Search Results


    Frequency distributions of intracellular staining of PDETB30‐OG488 in Caco‐2 cells. Cellular internalization examined in the presence of no inhibitor (a), chlorpromazine (b), filipin III (c), nystatin (d), wortmannin (e), amiloride (f), or 4 °C (g). Untreated (no PDETB30‐OG488) is shown in panel (h). Caco‐2 cells were preincubated with inhibitors for 30 min, exposed to 25 μg ml −1 PDETB30‐OG488 for 60 min, and imaged via ImageStream cytometry after 60 min further incubation. Histograms generated from image analysis of at least 500 cells

    Journal: Bioengineering & Translational Medicine

    Article Title: Uptake and function of membrane‐destabilizing cationic nanogels for intracellular drug delivery. Uptake and function of membrane‐destabilizing cationic nanogels for intracellular drug delivery

    doi: 10.1002/btm2.10120

    Figure Lengend Snippet: Frequency distributions of intracellular staining of PDETB30‐OG488 in Caco‐2 cells. Cellular internalization examined in the presence of no inhibitor (a), chlorpromazine (b), filipin III (c), nystatin (d), wortmannin (e), amiloride (f), or 4 °C (g). Untreated (no PDETB30‐OG488) is shown in panel (h). Caco‐2 cells were preincubated with inhibitors for 30 min, exposed to 25 μg ml −1 PDETB30‐OG488 for 60 min, and imaged via ImageStream cytometry after 60 min further incubation. Histograms generated from image analysis of at least 500 cells

    Article Snippet: Filipin III was purchased from Cayman Chemical (Ann Arbor, MI).

    Techniques: Staining, Cytometry, Incubation, Generated

    Additional NMJ images and quantifications of bouton number and bouton size. ( A ) Quantification of bouton number of NMJ4 from wild-type larvae fed with 0 mM (CT), 10 mM, 20 mM MβCD and 0 μg/ml (DMSO vehicle only), 25 μg/ml, 50 μg/ml filipin III. ( B and C ) Quantifications of muscle four surface area ( B ), relative bouton number and size normalized to muscle surface area ( C ) upon indicated treatments. n ≥ 8 larvae; ns, no significance, *p

    Journal: eLife

    Article Title: The glycosphingolipid MacCer promotes synaptic bouton formation in Drosophila by interacting with Wnt

    doi: 10.7554/eLife.38183

    Figure Lengend Snippet: Additional NMJ images and quantifications of bouton number and bouton size. ( A ) Quantification of bouton number of NMJ4 from wild-type larvae fed with 0 mM (CT), 10 mM, 20 mM MβCD and 0 μg/ml (DMSO vehicle only), 25 μg/ml, 50 μg/ml filipin III. ( B and C ) Quantifications of muscle four surface area ( B ), relative bouton number and size normalized to muscle surface area ( C ) upon indicated treatments. n ≥ 8 larvae; ns, no significance, *p

    Article Snippet: D,L-threo-PDMP (Matreya) and filipin III (Cayman) were dissolved in DMSO and then individually added to standard media at specific concentrations.

    Techniques:

    Syn acts on the content of plasma membrane cholesterol that affects calcium channel activation and neurotransmitter release. A , Bar chart showing cholesterol levels determined by quantitative fluorescent filipin staining. Cortical neurons incubated with

    Journal: The Journal of Neuroscience

    Article Title: Exogenous α-Synuclein Decreases Raft Partitioning of Cav2.2 Channels Inducing Dopamine Release

    doi: 10.1523/JNEUROSCI.0608-14.2014

    Figure Lengend Snippet: Syn acts on the content of plasma membrane cholesterol that affects calcium channel activation and neurotransmitter release. A , Bar chart showing cholesterol levels determined by quantitative fluorescent filipin staining. Cortical neurons incubated with

    Article Snippet: Filipin quantitative staining of fixed cells was performed accordingly to a modification of a commercially available kit (catalog #10009779; Cayman Chemicals).

    Techniques: Activation Assay, Staining, Incubation

    Long term HPβCD treatment increases cellular cholesterol. ( Top Row ) - Filipin staining shows HPβCD treatment, LF loading and the two combined each cause substantial cellular cholesterol increase relative to healthy cells. ( Bottom Row ) Cells with increased lysosome cholesterol composition are more resistant to apoptosis induced by the lysomotropic agent MSDH. Healthy confluent cells were killed by MSDH exposure (45 uM MSDH for 42 hours) while HPβCD treated cells and LF loaded cells survived with no signs of visual stress. Therefore, HPβCD and LF modulate lysosome membrane cholesterol composition in a manner consistent with that suggested by filipin staining.

    Journal: Scientific Reports

    Article Title: 2-Hydroxypropyl-beta-cyclodextrin (HPβCD) reduces age-related lipofuscin accumulation through a cholesterol-associated pathway

    doi: 10.1038/s41598-017-02387-8

    Figure Lengend Snippet: Long term HPβCD treatment increases cellular cholesterol. ( Top Row ) - Filipin staining shows HPβCD treatment, LF loading and the two combined each cause substantial cellular cholesterol increase relative to healthy cells. ( Bottom Row ) Cells with increased lysosome cholesterol composition are more resistant to apoptosis induced by the lysomotropic agent MSDH. Healthy confluent cells were killed by MSDH exposure (45 uM MSDH for 42 hours) while HPβCD treated cells and LF loaded cells survived with no signs of visual stress. Therefore, HPβCD and LF modulate lysosome membrane cholesterol composition in a manner consistent with that suggested by filipin staining.

    Article Snippet: Cholesterol detection with filipin and O-methyl-serine dodecylamide hydrochloride (MSDH) Filipin staining was performed using the Cholesterol Cell-Based Detection Assay Kit (Cayman Chemical 10009779) and protocol.

    Techniques: Staining