Structured Review

Carl Zeiss filipin
ABCG1-GFP traffics between the cell surface and late endosomes. ABCG1-GFP resides on the cell surface and intracellular vesicles ( a ; green ), vitally labeled with endocytosed Alexa594 dextran ( b ; red ), used as a marker for late endosomes/lysosomes. As seen in ( c ), the ABCG1 late endocytic vesicles are enriched with cholesterol as revealed by cholesterol-specific cytochemical <t>filipin</t> staining ( blue ). 3D reconstruction of the entire volume of an ABCG1-expressing cell shows ABCG1 in the cell surface ( d ; single frame from Movie 1). Rendering of the data set in ( d ) to reveal intracellular ABCG1 ( e ) demonstrates the colocalization of ABCG1 ( green ) in late endosomes, labeled as in ( b ) with fluorescent dextran ( red ), as yellow punctate structures in this merged image. Note the ABCG1 late endosomes/lysosomes localize in abundance in the perinuclear region as well as in peripheral locations close to the cell surface ( e ; single frame from Movie 2); ( f ) Time lapse confocal microscopy reveals trafficking of ABCG1-late endosomes between the perinuclear region and cell surface as well as contact of ABCG1-late endosomes with each other and the PM (Single frame from Movie 3); ( g ) 3D-Time lapse confocal microscopy shows extensive interaction of ABCG1 late endosomes with the cell surface (Single frame from Movie 4).
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1) Product Images from "Cellular Localization and Trafficking of the Human ABCG1 Transporter"

Article Title: Cellular Localization and Trafficking of the Human ABCG1 Transporter

Journal: Biology

doi: 10.3390/biology3040781

ABCG1-GFP traffics between the cell surface and late endosomes. ABCG1-GFP resides on the cell surface and intracellular vesicles ( a ; green ), vitally labeled with endocytosed Alexa594 dextran ( b ; red ), used as a marker for late endosomes/lysosomes. As seen in ( c ), the ABCG1 late endocytic vesicles are enriched with cholesterol as revealed by cholesterol-specific cytochemical filipin staining ( blue ). 3D reconstruction of the entire volume of an ABCG1-expressing cell shows ABCG1 in the cell surface ( d ; single frame from Movie 1). Rendering of the data set in ( d ) to reveal intracellular ABCG1 ( e ) demonstrates the colocalization of ABCG1 ( green ) in late endosomes, labeled as in ( b ) with fluorescent dextran ( red ), as yellow punctate structures in this merged image. Note the ABCG1 late endosomes/lysosomes localize in abundance in the perinuclear region as well as in peripheral locations close to the cell surface ( e ; single frame from Movie 2); ( f ) Time lapse confocal microscopy reveals trafficking of ABCG1-late endosomes between the perinuclear region and cell surface as well as contact of ABCG1-late endosomes with each other and the PM (Single frame from Movie 3); ( g ) 3D-Time lapse confocal microscopy shows extensive interaction of ABCG1 late endosomes with the cell surface (Single frame from Movie 4).
Figure Legend Snippet: ABCG1-GFP traffics between the cell surface and late endosomes. ABCG1-GFP resides on the cell surface and intracellular vesicles ( a ; green ), vitally labeled with endocytosed Alexa594 dextran ( b ; red ), used as a marker for late endosomes/lysosomes. As seen in ( c ), the ABCG1 late endocytic vesicles are enriched with cholesterol as revealed by cholesterol-specific cytochemical filipin staining ( blue ). 3D reconstruction of the entire volume of an ABCG1-expressing cell shows ABCG1 in the cell surface ( d ; single frame from Movie 1). Rendering of the data set in ( d ) to reveal intracellular ABCG1 ( e ) demonstrates the colocalization of ABCG1 ( green ) in late endosomes, labeled as in ( b ) with fluorescent dextran ( red ), as yellow punctate structures in this merged image. Note the ABCG1 late endosomes/lysosomes localize in abundance in the perinuclear region as well as in peripheral locations close to the cell surface ( e ; single frame from Movie 2); ( f ) Time lapse confocal microscopy reveals trafficking of ABCG1-late endosomes between the perinuclear region and cell surface as well as contact of ABCG1-late endosomes with each other and the PM (Single frame from Movie 3); ( g ) 3D-Time lapse confocal microscopy shows extensive interaction of ABCG1 late endosomes with the cell surface (Single frame from Movie 4).

Techniques Used: Labeling, Marker, Staining, Expressing, Confocal Microscopy

2) Product Images from "The AGC Kinase YpkA Regulates Sphingolipids Biosynthesis and Physically Interacts With SakA MAP Kinase in Aspergillus fumigatus"

Article Title: The AGC Kinase YpkA Regulates Sphingolipids Biosynthesis and Physically Interacts With SakA MAP Kinase in Aspergillus fumigatus

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2018.03347

The content and cellular distributions of ergosterol are abnormal under ypkA loss of function. (A) 1 × 10 3 conidia of wild-type strain and a cell amount of Δ ypkA mutant were inocul ated in liquid YG culture and incubated at 37°C for 120 h before ergosterol extraction and quantification by HPLC (see section “Materials and Methods” for details). (B) 1 × 10 7 conidia of both wild-type and niiA::ypkA strains were inoculated in liquid AMM supplemented with MN or AT and incubated at 37°C for 24 h before ergosterol extraction and quantification by HPLC. Experiments were performed in triplicate and the results are the mean ± SD. Results show the ergosterol concentration normalized by the mycelia dry weight. ∗ Statistically significant (Student’s t -test; p ≤ 0.05). (C) 1 × 10 5 conidia of niiA::ypkA strain were inoculated in liquid AMM supplemented with MN or AT and incubated at 37°C for 12 h. The germilings were stained with 25 μg/ml of filipin for 5 min, observed in a fluorescence microscope and photographed. Dashed bars = 20 μm. Solid bars = 10 μm.
Figure Legend Snippet: The content and cellular distributions of ergosterol are abnormal under ypkA loss of function. (A) 1 × 10 3 conidia of wild-type strain and a cell amount of Δ ypkA mutant were inocul ated in liquid YG culture and incubated at 37°C for 120 h before ergosterol extraction and quantification by HPLC (see section “Materials and Methods” for details). (B) 1 × 10 7 conidia of both wild-type and niiA::ypkA strains were inoculated in liquid AMM supplemented with MN or AT and incubated at 37°C for 24 h before ergosterol extraction and quantification by HPLC. Experiments were performed in triplicate and the results are the mean ± SD. Results show the ergosterol concentration normalized by the mycelia dry weight. ∗ Statistically significant (Student’s t -test; p ≤ 0.05). (C) 1 × 10 5 conidia of niiA::ypkA strain were inoculated in liquid AMM supplemented with MN or AT and incubated at 37°C for 12 h. The germilings were stained with 25 μg/ml of filipin for 5 min, observed in a fluorescence microscope and photographed. Dashed bars = 20 μm. Solid bars = 10 μm.

Techniques Used: Mutagenesis, Incubation, High Performance Liquid Chromatography, Concentration Assay, Staining, Fluorescence, Microscopy

3) Product Images from "A human neuronal model of Niemann Pick C disease developed from stem cells isolated from patient's skin"

Article Title: A human neuronal model of Niemann Pick C disease developed from stem cells isolated from patient's skin

Journal: Orphanet Journal of Rare Diseases

doi: 10.1186/1750-1172-8-34

Characterization of stem cell populations obtained from skin biopsies or already established skin fibroblast cultures. ( A - D ) Phase contrast images of hSKIN-MASC at the third passage in culture: A - B ) hSKIN-MASC obtained from skin biopsies of a healthy donor ( A ) and a NPC patient ( B ). C - D ) hSKIN-MASC obtained from already established skin fibroblasts from healthy donor ( C ) and NPC patient ( D ). ( E ) Surface immunophenotype: representative flow cytometry histograms of skin derived stem cell cultures. Plots show isotype control IgG-staining profile (green histograms) versus specific antibody staining (red histograms). ( F - H ) Pluripotent state specific transcription factor expression: representative fluorescence images of Oct-4 (green fluorescence; F ), Nanog (green fluorescence; G ) and Sox-2 expression (green fluorescence; H ) localized in the nuclei of skin derived stem cell cultures. Nuclei are depicted by the blue fluorescence of DAPI staining ( F-H ). I) Quantification of the percentage of cells expressing Oct4 (white bars), Nanog (black bars) and Sox-2 (gray bars) in hSKIN-MASC obtained both from healthy donors (CTRL, n = 3) and NPC (n = 3) skin biopsies or healthy donors (CTRL, n = 3) and NPC (n = 3) skin fibroblast cultures. At least 400 cells have been counted for each cell line. Data are presented as mean ± SD of 3 independent experiments. ( J - K ) Nestin expression (red fluorescence) in hSKIN-MASC obtained from a healthy donor ( J ) and a NPC patient ( K ). ( L - N ) Unesterified cholesterol accumulation: images obtained after performing the filipin staining (blue fluorescence) in hSKIN-MASC derived from a healthy donor skin biopsy ( L ), a NPC patient skin biopsy ( M) and a NPC skin fibroblast cell line ( N ) , respectively.
Figure Legend Snippet: Characterization of stem cell populations obtained from skin biopsies or already established skin fibroblast cultures. ( A - D ) Phase contrast images of hSKIN-MASC at the third passage in culture: A - B ) hSKIN-MASC obtained from skin biopsies of a healthy donor ( A ) and a NPC patient ( B ). C - D ) hSKIN-MASC obtained from already established skin fibroblasts from healthy donor ( C ) and NPC patient ( D ). ( E ) Surface immunophenotype: representative flow cytometry histograms of skin derived stem cell cultures. Plots show isotype control IgG-staining profile (green histograms) versus specific antibody staining (red histograms). ( F - H ) Pluripotent state specific transcription factor expression: representative fluorescence images of Oct-4 (green fluorescence; F ), Nanog (green fluorescence; G ) and Sox-2 expression (green fluorescence; H ) localized in the nuclei of skin derived stem cell cultures. Nuclei are depicted by the blue fluorescence of DAPI staining ( F-H ). I) Quantification of the percentage of cells expressing Oct4 (white bars), Nanog (black bars) and Sox-2 (gray bars) in hSKIN-MASC obtained both from healthy donors (CTRL, n = 3) and NPC (n = 3) skin biopsies or healthy donors (CTRL, n = 3) and NPC (n = 3) skin fibroblast cultures. At least 400 cells have been counted for each cell line. Data are presented as mean ± SD of 3 independent experiments. ( J - K ) Nestin expression (red fluorescence) in hSKIN-MASC obtained from a healthy donor ( J ) and a NPC patient ( K ). ( L - N ) Unesterified cholesterol accumulation: images obtained after performing the filipin staining (blue fluorescence) in hSKIN-MASC derived from a healthy donor skin biopsy ( L ), a NPC patient skin biopsy ( M) and a NPC skin fibroblast cell line ( N ) , respectively.

Techniques Used: Flow Cytometry, Cytometry, Derivative Assay, Staining, Expressing, Fluorescence

4) Product Images from "Role of Cathepsin D in U18666A-induced Neuronal Cell Death"

Article Title: Role of Cathepsin D in U18666A-induced Neuronal Cell Death

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M112.412460

Increased U18666A-induced neurotoxicity and cholesterol accumulation in mouse primary hippocampal neurons. After 5 days of plating, cultured neurons were treated with 0.1–50 μg/ml U18666A for 24 h ( A ) or with 5 μg/ml U18666A for 6–96 h ( B ). MTT values were significantly attenuated in concentration-dependent ( A ) and time-dependent ( B ) manners in U18666A-treated cultures compared with mock-treated cultures. C and D , increase in the number of Hoechst 33258-labeled pycnotic nuclei following treatment with 0.1–50 μg/ml U18666A for 24 h ( C ) or with 5 μg/ml U18666A for 6–96 h ( D ). E and F , presence of condensed and/or fragmented nuclei ( arrows ) in control and U18666A-treated neuronal cultures. G and H , Live/Dead assay showing calcein AM staining in living neurons ( green , G ; arrowheads ) and EthD-1-labeled dead neurons following treatment with 5 μg/ml U18666A for 24 h ( red , H ; arrows ). I and J represent cholesterol accumulation, as evident by filipin staining in control and U18666A-treated neurons. All results, which are presented as means ± S.E. ( error bars ), were obtained from three separate experiments, each performed in triplicate. UA , U18666A; Ctrl , control. Scale bar , 25 μm. *, p
Figure Legend Snippet: Increased U18666A-induced neurotoxicity and cholesterol accumulation in mouse primary hippocampal neurons. After 5 days of plating, cultured neurons were treated with 0.1–50 μg/ml U18666A for 24 h ( A ) or with 5 μg/ml U18666A for 6–96 h ( B ). MTT values were significantly attenuated in concentration-dependent ( A ) and time-dependent ( B ) manners in U18666A-treated cultures compared with mock-treated cultures. C and D , increase in the number of Hoechst 33258-labeled pycnotic nuclei following treatment with 0.1–50 μg/ml U18666A for 24 h ( C ) or with 5 μg/ml U18666A for 6–96 h ( D ). E and F , presence of condensed and/or fragmented nuclei ( arrows ) in control and U18666A-treated neuronal cultures. G and H , Live/Dead assay showing calcein AM staining in living neurons ( green , G ; arrowheads ) and EthD-1-labeled dead neurons following treatment with 5 μg/ml U18666A for 24 h ( red , H ; arrows ). I and J represent cholesterol accumulation, as evident by filipin staining in control and U18666A-treated neurons. All results, which are presented as means ± S.E. ( error bars ), were obtained from three separate experiments, each performed in triplicate. UA , U18666A; Ctrl , control. Scale bar , 25 μm. *, p

Techniques Used: Cell Culture, MTT Assay, Concentration Assay, Labeling, Live Dead Assay, Staining, Ethidium Homodimer Assay

5) Product Images from "Treatment of Human Fibroblasts Carrying NPC1 Missense Mutations with MG132 Leads to an Improvement of Intracellular Cholesterol Trafficking"

Article Title: Treatment of Human Fibroblasts Carrying NPC1 Missense Mutations with MG132 Leads to an Improvement of Intracellular Cholesterol Trafficking

Journal: JIMD Reports

doi: 10.1007/8904_2011_49

Immunofluorescence staining of GM1 and filipin staining of intracellular unesterified cholesterol in “responsive” NPC fibroblasts in the absence and presence of MG132. Under basal conditions a massive accumulation of cholesterol and GM1
Figure Legend Snippet: Immunofluorescence staining of GM1 and filipin staining of intracellular unesterified cholesterol in “responsive” NPC fibroblasts in the absence and presence of MG132. Under basal conditions a massive accumulation of cholesterol and GM1

Techniques Used: Immunofluorescence, Staining

Filipin staining of intracellular unesterified cholesterol. ( a ) Normal fibroblasts; ( b and d ) NPC fibroblasts treated for 10 days with vehicle showed a massive lysosomal accumulation of unesterified cholesterol; ( c and e ) NPC fibroblasts treated
Figure Legend Snippet: Filipin staining of intracellular unesterified cholesterol. ( a ) Normal fibroblasts; ( b and d ) NPC fibroblasts treated for 10 days with vehicle showed a massive lysosomal accumulation of unesterified cholesterol; ( c and e ) NPC fibroblasts treated

Techniques Used: Staining

6) Product Images from "Regulatory role of ?-arrestin-2 in cholesterol processing in cystic fibrosis epithelial cells"

Article Title: Regulatory role of ?-arrestin-2 in cholesterol processing in cystic fibrosis epithelial cells

Journal: Journal of Lipid Research

doi: 10.1194/jlr.M021972

Localization of cholesterol and βarr2 in the lysosomes in CF and βarr2-overexpressing cells. (A) Representative image of lysosomes (LysoTracker) and endogenous free cholesterol (filipin) in GFP-expressing control cells. (B) Representative
Figure Legend Snippet: Localization of cholesterol and βarr2 in the lysosomes in CF and βarr2-overexpressing cells. (A) Representative image of lysosomes (LysoTracker) and endogenous free cholesterol (filipin) in GFP-expressing control cells. (B) Representative

Techniques Used: Expressing

Correction of cholesterol accumulation in βarr2-overexpressing cells with Rp -cAMPS (50 μM). (A) Representative image of untreated GFP-expressing (control-GFP) cells stained for endogenous free cholesterol (filipin). (B) Representative
Figure Legend Snippet: Correction of cholesterol accumulation in βarr2-overexpressing cells with Rp -cAMPS (50 μM). (A) Representative image of untreated GFP-expressing (control-GFP) cells stained for endogenous free cholesterol (filipin). (B) Representative

Techniques Used: Expressing, Staining

Cholesterol accumulation in βarr2-expressing 9/9/HTEo− cells. (A) Representative images of GFP-expressing control cells stained for endogenous free cholesterol (filipin). (B) Representative images of GFP-tagged βarr2-expressing
Figure Legend Snippet: Cholesterol accumulation in βarr2-expressing 9/9/HTEo− cells. (A) Representative images of GFP-expressing control cells stained for endogenous free cholesterol (filipin). (B) Representative images of GFP-tagged βarr2-expressing

Techniques Used: Expressing, Staining

7) Product Images from "The AGC Kinase YpkA Regulates Sphingolipids Biosynthesis and Physically Interacts With SakA MAP Kinase in Aspergillus fumigatus"

Article Title: The AGC Kinase YpkA Regulates Sphingolipids Biosynthesis and Physically Interacts With SakA MAP Kinase in Aspergillus fumigatus

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2018.03347

The content and cellular distributions of ergosterol are abnormal under ypkA loss of function. (A) 1 × 10 3 conidia of wild-type strain and a cell amount of Δ ypkA mutant were inoculated in liquid YG culture and incubated at 37°C for 120 h before ergosterol extraction and quantification by HPLC (see section “Materials and Methods” for details). (B) 1 × 10 7 conidia of both wild-type and niiA::ypkA strains were inoculated in liquid AMM supplemented with MN or AT and incubated at 37°C for 24 h before ergosterol extraction and quantification by HPLC. Experiments were performed in triplicate and the results are the mean ± SD. Results show the ergosterol concentration normalized by the mycelia dry weight. ∗ Statistically significant (Student’s t -test; p ≤ 0.05). (C) 1 × 10 5 conidia of niiA::ypkA strain were inoculated in liquid AMM supplemented with MN or AT and incubated at 37°C for 12 h. The germilings were stained with 25 μg/ml of filipin for 5 min, observed in a fluorescence microscope and photographed. Dashed bars = 20 μm. Solid bars = 10 μm.
Figure Legend Snippet: The content and cellular distributions of ergosterol are abnormal under ypkA loss of function. (A) 1 × 10 3 conidia of wild-type strain and a cell amount of Δ ypkA mutant were inoculated in liquid YG culture and incubated at 37°C for 120 h before ergosterol extraction and quantification by HPLC (see section “Materials and Methods” for details). (B) 1 × 10 7 conidia of both wild-type and niiA::ypkA strains were inoculated in liquid AMM supplemented with MN or AT and incubated at 37°C for 24 h before ergosterol extraction and quantification by HPLC. Experiments were performed in triplicate and the results are the mean ± SD. Results show the ergosterol concentration normalized by the mycelia dry weight. ∗ Statistically significant (Student’s t -test; p ≤ 0.05). (C) 1 × 10 5 conidia of niiA::ypkA strain were inoculated in liquid AMM supplemented with MN or AT and incubated at 37°C for 12 h. The germilings were stained with 25 μg/ml of filipin for 5 min, observed in a fluorescence microscope and photographed. Dashed bars = 20 μm. Solid bars = 10 μm.

Techniques Used: Mutagenesis, Incubation, High Performance Liquid Chromatography, Concentration Assay, Staining, Fluorescence, Microscopy

8) Product Images from "Inhibition of angiogenesis by selective estrogen receptor modulators through blockade of cholesterol trafficking rather than estrogen receptor antagonism"

Article Title: Inhibition of angiogenesis by selective estrogen receptor modulators through blockade of cholesterol trafficking rather than estrogen receptor antagonism

Journal: Cancer letters

doi: 10.1016/j.canlet.2015.03.022

Effect of SERM on cholesterol trafficking in HUVEC. (A) HUVEC were treated with tamoxifen (TMX), toremifene (TRM), clomifene (CLM) and raloxifene (RLX) for 24 h, and intracellular cholesterol was visualized by filipin. (B) HUVEC were treated with 1 μM
Figure Legend Snippet: Effect of SERM on cholesterol trafficking in HUVEC. (A) HUVEC were treated with tamoxifen (TMX), toremifene (TRM), clomifene (CLM) and raloxifene (RLX) for 24 h, and intracellular cholesterol was visualized by filipin. (B) HUVEC were treated with 1 μM

Techniques Used:

9) Product Images from "Characterization of a Nonclathrin Endocytic Pathway: Membrane Cargo and Lipid Requirements D⃞"

Article Title: Characterization of a Nonclathrin Endocytic Pathway: Membrane Cargo and Lipid Requirements D⃞

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E04-02-0151

CD59 and cholesterol reach Rab5 compartment via Arf6-endosomes. HeLa cells were transfected with GFP-Rab5-Q79L alone (A and C) or with GFP-Rab5-Q79L and Arf6-Q67L (B and D). In A and B, cells were allowed to internalize phycoerythrin anti-CD59 antibody at 37°C for 1 h. In C and D, cells were fixed and stained with filipin. Bar, 10 μm.
Figure Legend Snippet: CD59 and cholesterol reach Rab5 compartment via Arf6-endosomes. HeLa cells were transfected with GFP-Rab5-Q79L alone (A and C) or with GFP-Rab5-Q79L and Arf6-Q67L (B and D). In A and B, cells were allowed to internalize phycoerythrin anti-CD59 antibody at 37°C for 1 h. In C and D, cells were fixed and stained with filipin. Bar, 10 μm.

Techniques Used: Transfection, Staining

Effect of endocytosis inhibitors on CD59 and MHCI internalization. (A and B) HeLa cells were transfected with FLAG-c-AP180 and internalization of anti-CD59 and anti-MHCI (A) or of anti-MHCI and Tfn (B) was assessed. (C and D) Untransfected HeLa cells were preincubated for 20 min at 37°C with DMEM containing 0.5% BSA in the absence (C) or presence (D) of filipin (12 μg/ml). Then, Cy5-anti MHCI, R-phycoerythrin-anti-CD59, and Alexa 488-Tfn (5 μg/ml) were added for a 20-min uptake. The cells were fixed and processed for immunofluorescence. All images were taken with identical acquisition parameters and the amount of internalized antibody for either c-AP180 (E) or filipin treatment (F) was measured and expressed as a percentage of that internalized in control cells (see MATERIALS AND METHODS). Bar, 10 μm (A and B) and 20 μm (C and D).
Figure Legend Snippet: Effect of endocytosis inhibitors on CD59 and MHCI internalization. (A and B) HeLa cells were transfected with FLAG-c-AP180 and internalization of anti-CD59 and anti-MHCI (A) or of anti-MHCI and Tfn (B) was assessed. (C and D) Untransfected HeLa cells were preincubated for 20 min at 37°C with DMEM containing 0.5% BSA in the absence (C) or presence (D) of filipin (12 μg/ml). Then, Cy5-anti MHCI, R-phycoerythrin-anti-CD59, and Alexa 488-Tfn (5 μg/ml) were added for a 20-min uptake. The cells were fixed and processed for immunofluorescence. All images were taken with identical acquisition parameters and the amount of internalized antibody for either c-AP180 (E) or filipin treatment (F) was measured and expressed as a percentage of that internalized in control cells (see MATERIALS AND METHODS). Bar, 10 μm (A and B) and 20 μm (C and D).

Techniques Used: Transfection, Immunofluorescence

Tac-GPI, accumulated in Arf6-Q67L–induced vacuoles, is within detergent-resistant membranes. Cells coexpressing Arf6-Q67L and Tac-GPI were allowed to internalize anti-Tac antibody at 37°C for 1 h, cooled on ice, acid stripped, and incubated 3 min with ice-cold PBS (A) or with PBS and 1% TX-100 (B). After fixation, Arf6 was stained with polyclonal antibody. Tac-GPI was visualized with 488 Alexa goat anti-mouse, followed by staining of the free cholesterol with filipin. Bar, 10 μm.
Figure Legend Snippet: Tac-GPI, accumulated in Arf6-Q67L–induced vacuoles, is within detergent-resistant membranes. Cells coexpressing Arf6-Q67L and Tac-GPI were allowed to internalize anti-Tac antibody at 37°C for 1 h, cooled on ice, acid stripped, and incubated 3 min with ice-cold PBS (A) or with PBS and 1% TX-100 (B). After fixation, Arf6 was stained with polyclonal antibody. Tac-GPI was visualized with 488 Alexa goat anti-mouse, followed by staining of the free cholesterol with filipin. Bar, 10 μm.

Techniques Used: Incubation, Staining

Accelerated endocytosis of GPI-AP and clathrin-independent cargo into Arf6-Q67L expressing cells. (A) Cells transfected with Tac-GPI, Tac or Tac-LL alone (black bars) or in combination with Arf6-wt (white bars), Arf6-Q67L (red bars), or Arf6-T27N (blue bars) were incubated with biotinylated anti-Tac for 20 min at 37°C. Cells expressing Tac-GPI, Tac, or Tac-LL also were treated with filipin before Tac-antibody internalization (green bars). The surface-bound antibody was then removed by a low pH solution rinse, and the sample was fixed and processed for CELISA (see MATERIALS AND METHODS). (B and C) Internalization of Tac-GPI and Tac (circles and triangles in B), and Tac-LL (squares in C) was measured in a continuous uptake by using CELISA. Cells expressing the cargo proteins alone (black symbols) or with Arf6-Q67L (red symbols) were incubated at 37°C with biotinylated anti-Tac for the indicated time and then processed as described above. The amount of internalized Tac antibody is expressed as a percentage of total cell-associated anti-Tac. This experiment was performed three times with similar results. Data are means of triplicates with SD.
Figure Legend Snippet: Accelerated endocytosis of GPI-AP and clathrin-independent cargo into Arf6-Q67L expressing cells. (A) Cells transfected with Tac-GPI, Tac or Tac-LL alone (black bars) or in combination with Arf6-wt (white bars), Arf6-Q67L (red bars), or Arf6-T27N (blue bars) were incubated with biotinylated anti-Tac for 20 min at 37°C. Cells expressing Tac-GPI, Tac, or Tac-LL also were treated with filipin before Tac-antibody internalization (green bars). The surface-bound antibody was then removed by a low pH solution rinse, and the sample was fixed and processed for CELISA (see MATERIALS AND METHODS). (B and C) Internalization of Tac-GPI and Tac (circles and triangles in B), and Tac-LL (squares in C) was measured in a continuous uptake by using CELISA. Cells expressing the cargo proteins alone (black symbols) or with Arf6-Q67L (red symbols) were incubated at 37°C with biotinylated anti-Tac for the indicated time and then processed as described above. The amount of internalized Tac antibody is expressed as a percentage of total cell-associated anti-Tac. This experiment was performed three times with similar results. Data are means of triplicates with SD.

Techniques Used: Expressing, Transfection, Incubation

CD59 and cholesterol accumulate in Arf6-Q67L-associated vacuolar endosomes, devoid of caveolin-1. (A) Cells expressing Arf6-Q67L were incubated with anti-CD59 at 37°C for 1 h, and surface antibody was removed and then processed for immunofluorescence. After fixation, free cholesterol was stained with filipin as described in MATERIALS AND METHODS. Note the redistribution and intensity of filipin in the transfected cells (encircled). (B) Cells expressing Arf6-Q67L were labeled with antibody to caveolin-1. Bar, 10 μm.
Figure Legend Snippet: CD59 and cholesterol accumulate in Arf6-Q67L-associated vacuolar endosomes, devoid of caveolin-1. (A) Cells expressing Arf6-Q67L were incubated with anti-CD59 at 37°C for 1 h, and surface antibody was removed and then processed for immunofluorescence. After fixation, free cholesterol was stained with filipin as described in MATERIALS AND METHODS. Note the redistribution and intensity of filipin in the transfected cells (encircled). (B) Cells expressing Arf6-Q67L were labeled with antibody to caveolin-1. Bar, 10 μm.

Techniques Used: Expressing, Incubation, Immunofluorescence, Staining, Transfection, Labeling

10) Product Images from "Endolysosome involvement in LDL cholesterol-induced Alzheimer's disease-like pathology in primary cultured neurons"

Article Title: Endolysosome involvement in LDL cholesterol-induced Alzheimer's disease-like pathology in primary cultured neurons

Journal: Life sciences

doi: 10.1016/j.lfs.2012.04.039

LDL cholesterol increased cholesterol accumulation in neurons. (A) Neurons treated with LDL cholesterol (50 µg/ml) for 3 days exhibited altered free cholesterol (filipin staining) distribution; cholesterol was distributed near the plasma membrane
Figure Legend Snippet: LDL cholesterol increased cholesterol accumulation in neurons. (A) Neurons treated with LDL cholesterol (50 µg/ml) for 3 days exhibited altered free cholesterol (filipin staining) distribution; cholesterol was distributed near the plasma membrane

Techniques Used: Staining

11) Product Images from "Regulatory role of ?-arrestin-2 in cholesterol processing in cystic fibrosis epithelial cells"

Article Title: Regulatory role of ?-arrestin-2 in cholesterol processing in cystic fibrosis epithelial cells

Journal: Journal of Lipid Research

doi: 10.1194/jlr.M021972

Localization of cholesterol and βarr2 in the lysosomes in CF and βarr2-overexpressing cells. (A) Representative image of lysosomes (LysoTracker) and endogenous free cholesterol (filipin) in GFP-expressing control cells. (B) Representative
Figure Legend Snippet: Localization of cholesterol and βarr2 in the lysosomes in CF and βarr2-overexpressing cells. (A) Representative image of lysosomes (LysoTracker) and endogenous free cholesterol (filipin) in GFP-expressing control cells. (B) Representative

Techniques Used: Expressing

Correction of cholesterol accumulation in βarr2-overexpressing cells with Rp -cAMPS (50 μM). (A) Representative image of untreated GFP-expressing (control-GFP) cells stained for endogenous free cholesterol (filipin). (B) Representative
Figure Legend Snippet: Correction of cholesterol accumulation in βarr2-overexpressing cells with Rp -cAMPS (50 μM). (A) Representative image of untreated GFP-expressing (control-GFP) cells stained for endogenous free cholesterol (filipin). (B) Representative

Techniques Used: Expressing, Staining

Cholesterol accumulation in βarr2-expressing 9/9/HTEo− cells. (A) Representative images of GFP-expressing control cells stained for endogenous free cholesterol (filipin). (B) Representative images of GFP-tagged βarr2-expressing
Figure Legend Snippet: Cholesterol accumulation in βarr2-expressing 9/9/HTEo− cells. (A) Representative images of GFP-expressing control cells stained for endogenous free cholesterol (filipin). (B) Representative images of GFP-tagged βarr2-expressing

Techniques Used: Expressing, Staining

12) Product Images from "The Human ABCG1 Transporter Mobilizes Plasma Membrane and Late Endosomal Non-Sphingomyelin-Associated-Cholesterol for Efflux and Esterification"

Article Title: The Human ABCG1 Transporter Mobilizes Plasma Membrane and Late Endosomal Non-Sphingomyelin-Associated-Cholesterol for Efflux and Esterification

Journal: Biology

doi: 10.3390/biology3040866

Myriocin reduces PM SM content and increases non-SM-associated cellular cholesterol pools and cholesterol efflux. ( A ) Myriocin reduces PM SM content. Control and ABCG1 cells pre-treated without myriocin ((−) MYR), or, with myriocin ((+) MYR) were immunostained with lysenin. ( B ) Myriocin increases cellular FC. Control and ABCG1 cells treated without ((−) MYR), or, with ((+) MYR) were stained with filipin. ( C ) Myriocin enhances CD-mediated PM FC efflux. Control ( black line ) and ABCG1 ( green line ) cells labeled with 3 H-FC were pre-treated without or with myr ( solid and dashed lines , respectively), and the percent of total cellular FC efflux CD (1 h at 4 °C) was determined. ( D ) Myriocin enhances CD-mediated cellular FC efflux. Control ( black ) and ABCG1 ( green ) cells labeled with 3 H-FC were pre-treated without or with myr ( solid and dashed lines , respectively), and the percent of total cellular FC efflux to CD (10 min at 37 °C) was determined. Myriocin enhances FC efflux to PC liposomes ( E ) and to HDL ( F ). ( E , F) : Black line , dashed black line : untreated, and myr-treated control cells, respectively; green line , dashed green line : untreated, and myr-treated ABCG1 cells, respectively. Dotted black and green lines represent difference curves for control and ABCG1 cells with myriocin treatment, respectively. All values are expressed as mean ± S.D. Data shown is representative of at least three replicate experiments. Two-way ANOVA analyses using multiple comparisons revealed that in ( C ), all values are significantly different ( p
Figure Legend Snippet: Myriocin reduces PM SM content and increases non-SM-associated cellular cholesterol pools and cholesterol efflux. ( A ) Myriocin reduces PM SM content. Control and ABCG1 cells pre-treated without myriocin ((−) MYR), or, with myriocin ((+) MYR) were immunostained with lysenin. ( B ) Myriocin increases cellular FC. Control and ABCG1 cells treated without ((−) MYR), or, with ((+) MYR) were stained with filipin. ( C ) Myriocin enhances CD-mediated PM FC efflux. Control ( black line ) and ABCG1 ( green line ) cells labeled with 3 H-FC were pre-treated without or with myr ( solid and dashed lines , respectively), and the percent of total cellular FC efflux CD (1 h at 4 °C) was determined. ( D ) Myriocin enhances CD-mediated cellular FC efflux. Control ( black ) and ABCG1 ( green ) cells labeled with 3 H-FC were pre-treated without or with myr ( solid and dashed lines , respectively), and the percent of total cellular FC efflux to CD (10 min at 37 °C) was determined. Myriocin enhances FC efflux to PC liposomes ( E ) and to HDL ( F ). ( E , F) : Black line , dashed black line : untreated, and myr-treated control cells, respectively; green line , dashed green line : untreated, and myr-treated ABCG1 cells, respectively. Dotted black and green lines represent difference curves for control and ABCG1 cells with myriocin treatment, respectively. All values are expressed as mean ± S.D. Data shown is representative of at least three replicate experiments. Two-way ANOVA analyses using multiple comparisons revealed that in ( C ), all values are significantly different ( p

Techniques Used: Staining, Labeling

ABCG1-GFP alters cholesterol detergent solubility. ( A ) Control and ABCG1 cells were incubated with PBS or SMase, washed, fixed and immunostained with lysenin as described in “ Experimental Section ”. ( B ) Control and ABCG1 cells were incubated with cold PBS or 1% TX-100 detergent for 30 min, washed, fixed and immunostained with lysenin as described in “ Experimental Section ”. ( C ) Control and ABCG1 cells pre-treated with or without SMase, were extracted with 1% TX-100 on ice for 30 min, fixed, filipin stained, and imaged by confocal microscopy as described in “ Experimental Section .” ( D ) Higher magnification of filipin-stained AGCG1 cells clearly reveals that LE FC (a) is removed by TX-100 (b). Enlargements of the boxed regions in (a) and (b), shown in (c) and (d), respectively, reveal that TX-100 treatment depleted FC from PM domains ( arrowheads ).
Figure Legend Snippet: ABCG1-GFP alters cholesterol detergent solubility. ( A ) Control and ABCG1 cells were incubated with PBS or SMase, washed, fixed and immunostained with lysenin as described in “ Experimental Section ”. ( B ) Control and ABCG1 cells were incubated with cold PBS or 1% TX-100 detergent for 30 min, washed, fixed and immunostained with lysenin as described in “ Experimental Section ”. ( C ) Control and ABCG1 cells pre-treated with or without SMase, were extracted with 1% TX-100 on ice for 30 min, fixed, filipin stained, and imaged by confocal microscopy as described in “ Experimental Section .” ( D ) Higher magnification of filipin-stained AGCG1 cells clearly reveals that LE FC (a) is removed by TX-100 (b). Enlargements of the boxed regions in (a) and (b), shown in (c) and (d), respectively, reveal that TX-100 treatment depleted FC from PM domains ( arrowheads ).

Techniques Used: Solubility, Incubation, Staining, Confocal Microscopy

ABCG1-GFP expression increases efflux of SMase-mobilized cholesterol from the PM and LE. ( A ) Cold CD depletes excess PM FC from living ABCG1 cells. Control (a–c), and ABCG1 (d–f) cells pre-treated without (a,b,d,e), or, with SMase (c,f), were FC-depleted using 20 mM CD for 1h at 4 °C (b,c,e,f), then stained with filipin. ( B ) ABCG1 increases PM non-SM-associated-FC. Control ( black ) and ABCG1 ( green ) cells labeled with 3 H-FC were incubated in the absence or presence of SMase (solid and dashed lines, respectively) and, the percent of total cellular FC effluxed to CD for 1 h at 4 °C was determined. ( C ) Warm CD depletes excess ABCG1-induced PM and LE FC. ( C ) Control (a–c), and ABCG1 (d–f) cells pre-treated without (a,b,d,e), or, with SMase (c,f), were incubated with 5 mM CD for 10 min at 37 °C. ( D ) ABCG1 increases non-SM-associated cellular FC. Control ( black ) and ABCG1 ( green ) cells labeled with 3 H-FC were incubated in the absence or presence of SMase (solid and dashed lines, respectively) and, the percent of total cellular FC effluxed to CD for 10 min at 37 °C was determined. ( E ) ABCG1 enhances efflux of SMase-mobilized FC to liposomes and ( F ) HDL. Control ( black ) and ABCG1 ( green ) cells labeled with 3 H-FC were incubated in the absence or presence of SMase (solid and dashed lines, respectively) for 4 h and, the percent of total cellular FC effluxed to liposomes or HDL was determined. For ( B , D ) PM SM-associated FC is represented by the difference curves (percent FC efflux of (SMase-treated cells)—(non-treated cells)); black and green dotted lines, control and ABCG1 cells, respectively). All values are expressed as mean + S.D. Data shown is representative of at least three replicate experiments Two-way ANOVA analyses using multiple comparisons revealed that ABCG1 expression and SMase treatment significantly increased the percent CD-mediated cholesterol efflux in ( B ) and ( D ) p
Figure Legend Snippet: ABCG1-GFP expression increases efflux of SMase-mobilized cholesterol from the PM and LE. ( A ) Cold CD depletes excess PM FC from living ABCG1 cells. Control (a–c), and ABCG1 (d–f) cells pre-treated without (a,b,d,e), or, with SMase (c,f), were FC-depleted using 20 mM CD for 1h at 4 °C (b,c,e,f), then stained with filipin. ( B ) ABCG1 increases PM non-SM-associated-FC. Control ( black ) and ABCG1 ( green ) cells labeled with 3 H-FC were incubated in the absence or presence of SMase (solid and dashed lines, respectively) and, the percent of total cellular FC effluxed to CD for 1 h at 4 °C was determined. ( C ) Warm CD depletes excess ABCG1-induced PM and LE FC. ( C ) Control (a–c), and ABCG1 (d–f) cells pre-treated without (a,b,d,e), or, with SMase (c,f), were incubated with 5 mM CD for 10 min at 37 °C. ( D ) ABCG1 increases non-SM-associated cellular FC. Control ( black ) and ABCG1 ( green ) cells labeled with 3 H-FC were incubated in the absence or presence of SMase (solid and dashed lines, respectively) and, the percent of total cellular FC effluxed to CD for 10 min at 37 °C was determined. ( E ) ABCG1 enhances efflux of SMase-mobilized FC to liposomes and ( F ) HDL. Control ( black ) and ABCG1 ( green ) cells labeled with 3 H-FC were incubated in the absence or presence of SMase (solid and dashed lines, respectively) for 4 h and, the percent of total cellular FC effluxed to liposomes or HDL was determined. For ( B , D ) PM SM-associated FC is represented by the difference curves (percent FC efflux of (SMase-treated cells)—(non-treated cells)); black and green dotted lines, control and ABCG1 cells, respectively). All values are expressed as mean + S.D. Data shown is representative of at least three replicate experiments Two-way ANOVA analyses using multiple comparisons revealed that ABCG1 expression and SMase treatment significantly increased the percent CD-mediated cholesterol efflux in ( B ) and ( D ) p

Techniques Used: Expressing, Staining, Labeling, Incubation

( A ) ABCG1-GFP expression increases the amount of rapidly recycling, non-SM-associated cholesterol at the PM and LE. Cells were labeled with filipin, BODIPY-SM and Alexa594-CtxB, and imaged as described in “ Experimental Section .” (a,f: GFP fluorescence; b,g: filipin; c,d,h,i; BODIPY-SM fluorescence). ( B ) ABCG1 expression does not alter SM-specific cytochemical lysenin staining at the PM. Note similar lysenin PM staining in single confocal image slices (a, b) as well as in 3D maximum projection images (c, d) of control and ABCG1 cells, respectively. ( C ) Cell surface GM 1 content and trafficking is not altered by ABCG1. The amount and distribution of fluorescent-tagged CtxB in living control (a, c) and ABCG1 cells (b, d) after labeling in the cold (a, b) and incubation for 60 min at 37 °C (c, d) is similar. Note that the imaging conditions in (a) and (f) were optimized for GFP fluorescence. The GFP fluorescence was exceeding low compared to the “green” BODIPY-SM signal and, the contribution of GFP to the “green” fluorescence in (c), (d), (h) and, (f) was essentially eliminated by adjusting the gain such that the GFP fluorescent signal from ABCG1 cells that were not labeled with BODIPY-SM was essentially eliminated ( Supplemental Figure 1 ).
Figure Legend Snippet: ( A ) ABCG1-GFP expression increases the amount of rapidly recycling, non-SM-associated cholesterol at the PM and LE. Cells were labeled with filipin, BODIPY-SM and Alexa594-CtxB, and imaged as described in “ Experimental Section .” (a,f: GFP fluorescence; b,g: filipin; c,d,h,i; BODIPY-SM fluorescence). ( B ) ABCG1 expression does not alter SM-specific cytochemical lysenin staining at the PM. Note similar lysenin PM staining in single confocal image slices (a, b) as well as in 3D maximum projection images (c, d) of control and ABCG1 cells, respectively. ( C ) Cell surface GM 1 content and trafficking is not altered by ABCG1. The amount and distribution of fluorescent-tagged CtxB in living control (a, c) and ABCG1 cells (b, d) after labeling in the cold (a, b) and incubation for 60 min at 37 °C (c, d) is similar. Note that the imaging conditions in (a) and (f) were optimized for GFP fluorescence. The GFP fluorescence was exceeding low compared to the “green” BODIPY-SM signal and, the contribution of GFP to the “green” fluorescence in (c), (d), (h) and, (f) was essentially eliminated by adjusting the gain such that the GFP fluorescent signal from ABCG1 cells that were not labeled with BODIPY-SM was essentially eliminated ( Supplemental Figure 1 ).

Techniques Used: Expressing, Labeling, Fluorescence, Staining, Incubation, Imaging

13) Product Images from "Transient Hepatic Overexpression of Insulin-Like Growth Factor 2 Induces Free Cholesterol and Lipid Droplet Formation"

Article Title: Transient Hepatic Overexpression of Insulin-Like Growth Factor 2 Induces Free Cholesterol and Lipid Droplet Formation

Journal: Frontiers in Physiology

doi: 10.3389/fphys.2016.00147

IGF2 induces lipid droplet formation but has no influence on lipid droplet size. (A) Representative LD540 fluorescence staining for lipid droplets (LD, yellow droplets) in animals injected with Igf2 plasmid (Igf2) compared to the control plasmid (co), nuclei were stained with DAPI (blue) (scale bar: 20 μm) (upper panel). Five randomly selected pictures per sample were collected and analyzed with ImageJ. Counted LDs were normalized to the nuclei count (lower panel, left ). Total LD area was normalized to the nuclei count (lower panel, middle ). Total LD size was estimated as the mean area of all counted LDs (lower panel, right ) (co, n = 10; Igf2, n = 11, independent two-sample t -test). (B) Representative filipin fluorescence staining for free cholesterol (black droplets) in Igf2 and co animals (co, n = 10; Igf2, n = 11, arrows designate examples for free cholesterol). (C) Levels of Hmgcr mRNA, real-time RT-PCR (co, n = 10; Igf2, n = 11, Wilcoxon rank sum test). Data were normalized to 18S mRNA and co. All results are presented as mean ± SEM.
Figure Legend Snippet: IGF2 induces lipid droplet formation but has no influence on lipid droplet size. (A) Representative LD540 fluorescence staining for lipid droplets (LD, yellow droplets) in animals injected with Igf2 plasmid (Igf2) compared to the control plasmid (co), nuclei were stained with DAPI (blue) (scale bar: 20 μm) (upper panel). Five randomly selected pictures per sample were collected and analyzed with ImageJ. Counted LDs were normalized to the nuclei count (lower panel, left ). Total LD area was normalized to the nuclei count (lower panel, middle ). Total LD size was estimated as the mean area of all counted LDs (lower panel, right ) (co, n = 10; Igf2, n = 11, independent two-sample t -test). (B) Representative filipin fluorescence staining for free cholesterol (black droplets) in Igf2 and co animals (co, n = 10; Igf2, n = 11, arrows designate examples for free cholesterol). (C) Levels of Hmgcr mRNA, real-time RT-PCR (co, n = 10; Igf2, n = 11, Wilcoxon rank sum test). Data were normalized to 18S mRNA and co. All results are presented as mean ± SEM.

Techniques Used: Fluorescence, Staining, Injection, Plasmid Preparation, Quantitative RT-PCR

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Concentration Assay:

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Incubation:

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Selection:

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Cell Culture:

Article Title: Role of Cathepsin D in U18666A-induced Neuronal Cell Death
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Confocal Microscopy:

Article Title: Cellular Localization and Trafficking of the Human ABCG1 Transporter
Article Snippet: .. Sucrosome formation was induced by incubation of cells grown in AMEM (Life Technologies, Inc.) medium, supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 IU/mL of penicillin, 100 µg/mL streptomycin, and 100 µg/mL G418 containing 60 mM sucrose in for 24 h. Confocal Microscopy—Laser scanning confocal microscopy to monitor GFP, filipin and Alexa 568 and Alexa 594 fluorescence, was performed using a Zeiss 510 LSCM. .. Time-lapse fluorescence microscopy and 3D imaging were performed as previously described [ ].

Article Title: Endolysosome involvement in LDL cholesterol-induced Alzheimer's disease-like pathology in primary cultured neurons
Article Snippet: Neurons stained with filipin for cholesterol were examined by fluorescence microscopy (Zeiss). .. Neurons stained with filipin for cholesterol were examined by fluorescence microscopy (Zeiss).

Article Title: The ER membrane protein complex promotes biogenesis of sterol-related enzymes maintaining cholesterol homeostasis
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Staining:

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Article Snippet: .. After washing them with PBS, the cells were incubated with 1.5 mg of glycine/ml PBS for 10 min, stained with filipin (0.05 mg/ml, in PBS 10% FCS) for 2 h and examined using a Zeiss fluorescence microscope. ..

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Article Snippet: Paragraph title: Staining and Microscopy ... Filipin and GFP were visualized using 49 and 38 HE filter sets (Carl Zeiss), respectively, and 100× magnification oil immersion objective (Plan-Apochromat; NA 1.4).

Article Title: Characterization of a Nonclathrin Endocytic Pathway: Membrane Cargo and Lipid Requirements D⃞
Article Snippet: Cells were stained with filipin as described previously ( ). .. Images of filipin were immediately captured on a charge-coupled device camera attached to an epifluorescence photomicroscope (Carl Zeiss) with a 63×/1.4 Plan Apo chromate objective, which has transmittance in the UV region.

Article Title: A human neuronal model of Niemann Pick C disease developed from stem cells isolated from patient's skin
Article Snippet: .. After washing them with PBS, the cells were incubated with 1.5 mg of glycine/ml PBS for 10 minutes, stained with filipin (0.05 mg/ml, in PBS 10% FCS) for 2 hours and examined using a Zeiss fluorescence microscope. .. Periodic acid Schiff staining (PAS) PAS was employed to detect glycogen accumulation.

Article Title: Role of Cathepsin D in U18666A-induced Neuronal Cell Death
Article Snippet: .. To determine cholesterol accumulation, control and U18666A-treated hippocampal cultured neurons or N2a cells were incubated in the dark with 125 μg/ml filipin in PBS for 1 h. Stained sections were examined using a Zeiss Axioskop-2 microscope. .. For Western blotting, control and drug-treated cells from different experimental paradigms were rinsed with cold TBS and then harvested in radioimmunoprecipitation assay buffer (TBS containing 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, and 10% glycerol with inhibitors 50 m m NaF, 1 m m NaVO3 , 10 μg/ml aprotinin, and 10 μg/ml leupeptin).

Article Title: Endolysosome involvement in LDL cholesterol-induced Alzheimer's disease-like pathology in primary cultured neurons
Article Snippet: .. Neurons stained with filipin for cholesterol were examined by fluorescence microscopy (Zeiss). .. To further determine the specificity of LDL cholesterol staining and its intracellular localization, we co-stained with lysoTracker dye (Invitrogen) and Dil-labeled LDL cholesterol (Kalein Biomecidal).

Article Title: Regulatory role of ?-arrestin-2 in cholesterol processing in cystic fibrosis epithelial cells
Article Snippet: Paragraph title: LysoTracker staining/filipin costaining ... Cells were visualized in the 595–605 nm range for the LysoTracker or the UV range for the filipin using a wide-field microscope on a Zeiss Axiovert 200 and Metamorph software.

Article Title: The ER membrane protein complex promotes biogenesis of sterol-related enzymes maintaining cholesterol homeostasis
Article Snippet: For Filipin III staining, after fixation with 4% PFA, cells were rinsed three times with PBS before quenching residual PFA with 1.5 mg/ml glycine plus PBS (10 min). .. Antibody-bound proteins were visualised at an excitation wavelength of 488 nm and DAPI and Filipin III were imaged at 405 nm using an LSM 710 (Zeiss, Oberkochen, Germany) confocal microscope.

Article Title: Inhibition of angiogenesis by selective estrogen receptor modulators through blockade of cholesterol trafficking rather than estrogen receptor antagonism
Article Snippet: For co-staining of proteins and cholesterol, HUVEC (1 × 104 cells/well) grown in a Nunc Lab-Tek II 8-Chamber Slide were treated with compounds for 24 h, fixed with 4% paraformaldehyde for 20 min at room temperature and stained with filipin (50 μg/ml) for 1 h at room temperature. .. Cells were then incubated with secondary antibodies in PBS with 50 μg/ml filipin, 0.05% saponin and 5% BSA at room temperature for 1 h. Cells were washed with PBS, mounted with Immu-mount, and observed under a Zeiss 510 Meta multiphoton confocal microscope.

Software:

Article Title: Cellular Localization and Trafficking of the Human ABCG1 Transporter
Article Snippet: Sucrosome formation was induced by incubation of cells grown in AMEM (Life Technologies, Inc.) medium, supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 IU/mL of penicillin, 100 µg/mL streptomycin, and 100 µg/mL G418 containing 60 mM sucrose in for 24 h. Confocal Microscopy—Laser scanning confocal microscopy to monitor GFP, filipin and Alexa 568 and Alexa 594 fluorescence, was performed using a Zeiss 510 LSCM. .. Time-lapse movie were made using Imaris software.

Article Title: The AGC Kinase YpkA Regulates Sphingolipids Biosynthesis and Physically Interacts With SakA MAP Kinase in Aspergillus fumigatus
Article Snippet: Filipin and GFP were visualized using 49 and 38 HE filter sets (Carl Zeiss), respectively, and 100× magnification oil immersion objective (Plan-Apochromat; NA 1.4). .. DIC (Differential Interference Contrast) and fluorescent images were captured with an AxioCam camera (Carl Zeiss) and processed using AxioVision software.

Article Title: Regulatory role of ?-arrestin-2 in cholesterol processing in cystic fibrosis epithelial cells
Article Snippet: .. Cells were visualized in the UV range for the filipin on a Zeiss Axiovert 200 and Metamorph software. .. NPC1-mCherry constructs were obtained from Dr. Kevin Vaughan (University of Notre Dame, Notre Dame, IN).

Article Title: Regulatory role of ?-arrestin-2 in cholesterol processing in cystic fibrosis epithelial cells
Article Snippet: .. Cells were visualized in the 595–605 nm range for the LysoTracker or the UV range for the filipin using a wide-field microscope on a Zeiss Axiovert 200 and Metamorph software. .. The βarr2-shRNA-expressing vectors and respective negative control vector were obtained from Qiagen (Germantown, MD).

Article Title: The ER membrane protein complex promotes biogenesis of sterol-related enzymes maintaining cholesterol homeostasis
Article Snippet: Antibody-bound proteins were visualised at an excitation wavelength of 488 nm and DAPI and Filipin III were imaged at 405 nm using an LSM 710 (Zeiss, Oberkochen, Germany) confocal microscope. .. Images were processed using ImageJ and Adobe Illustrator software.

Immunofluorescence:

Article Title: The ER membrane protein complex promotes biogenesis of sterol-related enzymes maintaining cholesterol homeostasis
Article Snippet: Paragraph title: Indirect immunofluorescence confocal microscopy ... Antibody-bound proteins were visualised at an excitation wavelength of 488 nm and DAPI and Filipin III were imaged at 405 nm using an LSM 710 (Zeiss, Oberkochen, Germany) confocal microscope.

Article Title: Inhibition of angiogenesis by selective estrogen receptor modulators through blockade of cholesterol trafficking rather than estrogen receptor antagonism
Article Snippet: Paragraph title: Immunofluorescence imaging ... Cells were then incubated with secondary antibodies in PBS with 50 μg/ml filipin, 0.05% saponin and 5% BSA at room temperature for 1 h. Cells were washed with PBS, mounted with Immu-mount, and observed under a Zeiss 510 Meta multiphoton confocal microscope.

Blocking Assay:

Article Title: The ER membrane protein complex promotes biogenesis of sterol-related enzymes maintaining cholesterol homeostasis
Article Snippet: Primary antibody [anti-SQS rabbit mAb, ab195046 (Abcam), 1:200] in blocking buffer was added (60 min), the coverslips were rinsed twice with PBS and incubated in the dark (60 min) with Alexa Fluor 488-conjugated secondary antibody (1:400 in blocking buffer). .. Antibody-bound proteins were visualised at an excitation wavelength of 488 nm and DAPI and Filipin III were imaged at 405 nm using an LSM 710 (Zeiss, Oberkochen, Germany) confocal microscope.

Article Title: Inhibition of angiogenesis by selective estrogen receptor modulators through blockade of cholesterol trafficking rather than estrogen receptor antagonism
Article Snippet: Cells were then incubated with secondary antibodies in PBS with 50 μg/ml filipin, 0.05% saponin and 5% BSA at room temperature for 1 h. Cells were washed with PBS, mounted with Immu-mount, and observed under a Zeiss 510 Meta multiphoton confocal microscope. .. For general immunofluorescence, cells were fixed with 4% paraformaldehyde for 10 min, permeabilized with 0.5% Triton X-100 for 10 min and washed with PBS prior to blocking in 1% BSA in PBS containing 0.1% tween 20 (PBST) for 1 h. Cells were then incubated with primary antibodies including anti-VEGFR2 (Cell Signaling Technology, Danvers, MA), anti-LAMP1 (SantaCruz Biotechnology, Santa Cruz, CA), anti-mTOR (Cell Signaling Technology) and anti-GM130 (BD Biosciences, San Jose, CA) in the blocking solution overnight at 4 °C, and then incubated with secondary antibodies conjugated with Alexa-Fluo488 or Alexa-Fluo594 for 1 h. The cellular nuclei were stained with 4’, 6-diamidino-2-phenylindole (DAPI) and actin cytoskeleton was stained with rhodamine-phalloidin (Life Technologies).

Imaging:

Article Title: Cellular Localization and Trafficking of the Human ABCG1 Transporter
Article Snippet: Sucrosome formation was induced by incubation of cells grown in AMEM (Life Technologies, Inc.) medium, supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 IU/mL of penicillin, 100 µg/mL streptomycin, and 100 µg/mL G418 containing 60 mM sucrose in for 24 h. Confocal Microscopy—Laser scanning confocal microscopy to monitor GFP, filipin and Alexa 568 and Alexa 594 fluorescence, was performed using a Zeiss 510 LSCM. .. Time-lapse fluorescence microscopy and 3D imaging were performed as previously described [ ].

Article Title: Inhibition of angiogenesis by selective estrogen receptor modulators through blockade of cholesterol trafficking rather than estrogen receptor antagonism
Article Snippet: Paragraph title: Immunofluorescence imaging ... Cells were then incubated with secondary antibodies in PBS with 50 μg/ml filipin, 0.05% saponin and 5% BSA at room temperature for 1 h. Cells were washed with PBS, mounted with Immu-mount, and observed under a Zeiss 510 Meta multiphoton confocal microscope.

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    Carl Zeiss filipin
    ABCG1-GFP traffics between the cell surface and late endosomes. ABCG1-GFP resides on the cell surface and intracellular vesicles ( a ; green ), vitally labeled with endocytosed Alexa594 dextran ( b ; red ), used as a marker for late endosomes/lysosomes. As seen in ( c ), the ABCG1 late endocytic vesicles are enriched with cholesterol as revealed by cholesterol-specific cytochemical <t>filipin</t> staining ( blue ). 3D reconstruction of the entire volume of an ABCG1-expressing cell shows ABCG1 in the cell surface ( d ; single frame from Movie 1). Rendering of the data set in ( d ) to reveal intracellular ABCG1 ( e ) demonstrates the colocalization of ABCG1 ( green ) in late endosomes, labeled as in ( b ) with fluorescent dextran ( red ), as yellow punctate structures in this merged image. Note the ABCG1 late endosomes/lysosomes localize in abundance in the perinuclear region as well as in peripheral locations close to the cell surface ( e ; single frame from Movie 2); ( f ) Time lapse confocal microscopy reveals trafficking of ABCG1-late endosomes between the perinuclear region and cell surface as well as contact of ABCG1-late endosomes with each other and the PM (Single frame from Movie 3); ( g ) 3D-Time lapse confocal microscopy shows extensive interaction of ABCG1 late endosomes with the cell surface (Single frame from Movie 4).
    Filipin, supplied by Carl Zeiss, used in various techniques. Bioz Stars score: 91/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/filipin/product/Carl Zeiss
    Average 91 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    filipin - by Bioz Stars, 2020-04
    91/100 stars
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    86
    Carl Zeiss filipin dye
    Fibroblast cell culture from the conjunctival biopsy of the proband. A : Fibroblastic cellular outgrowth from the conjunctival biopsy tissue. B : Following one week of cell culture, cellular confluence of fibroblasts is achieved. C : Following the addition of <t>filipin</t> stain, there is no evidence of cholesterol in the cultured conjunctival fibroblasts. D : Cells cultured with the addition of low density lipoprotein (LDL), acted as a positive control showing evidence of cholesterol with filipin staining. (Bar=100 microns).
    Filipin Dye, supplied by Carl Zeiss, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/filipin dye/product/Carl Zeiss
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    filipin dye - by Bioz Stars, 2020-04
    86/100 stars
      Buy from Supplier

    Image Search Results


    ABCG1-GFP traffics between the cell surface and late endosomes. ABCG1-GFP resides on the cell surface and intracellular vesicles ( a ; green ), vitally labeled with endocytosed Alexa594 dextran ( b ; red ), used as a marker for late endosomes/lysosomes. As seen in ( c ), the ABCG1 late endocytic vesicles are enriched with cholesterol as revealed by cholesterol-specific cytochemical filipin staining ( blue ). 3D reconstruction of the entire volume of an ABCG1-expressing cell shows ABCG1 in the cell surface ( d ; single frame from Movie 1). Rendering of the data set in ( d ) to reveal intracellular ABCG1 ( e ) demonstrates the colocalization of ABCG1 ( green ) in late endosomes, labeled as in ( b ) with fluorescent dextran ( red ), as yellow punctate structures in this merged image. Note the ABCG1 late endosomes/lysosomes localize in abundance in the perinuclear region as well as in peripheral locations close to the cell surface ( e ; single frame from Movie 2); ( f ) Time lapse confocal microscopy reveals trafficking of ABCG1-late endosomes between the perinuclear region and cell surface as well as contact of ABCG1-late endosomes with each other and the PM (Single frame from Movie 3); ( g ) 3D-Time lapse confocal microscopy shows extensive interaction of ABCG1 late endosomes with the cell surface (Single frame from Movie 4).

    Journal: Biology

    Article Title: Cellular Localization and Trafficking of the Human ABCG1 Transporter

    doi: 10.3390/biology3040781

    Figure Lengend Snippet: ABCG1-GFP traffics between the cell surface and late endosomes. ABCG1-GFP resides on the cell surface and intracellular vesicles ( a ; green ), vitally labeled with endocytosed Alexa594 dextran ( b ; red ), used as a marker for late endosomes/lysosomes. As seen in ( c ), the ABCG1 late endocytic vesicles are enriched with cholesterol as revealed by cholesterol-specific cytochemical filipin staining ( blue ). 3D reconstruction of the entire volume of an ABCG1-expressing cell shows ABCG1 in the cell surface ( d ; single frame from Movie 1). Rendering of the data set in ( d ) to reveal intracellular ABCG1 ( e ) demonstrates the colocalization of ABCG1 ( green ) in late endosomes, labeled as in ( b ) with fluorescent dextran ( red ), as yellow punctate structures in this merged image. Note the ABCG1 late endosomes/lysosomes localize in abundance in the perinuclear region as well as in peripheral locations close to the cell surface ( e ; single frame from Movie 2); ( f ) Time lapse confocal microscopy reveals trafficking of ABCG1-late endosomes between the perinuclear region and cell surface as well as contact of ABCG1-late endosomes with each other and the PM (Single frame from Movie 3); ( g ) 3D-Time lapse confocal microscopy shows extensive interaction of ABCG1 late endosomes with the cell surface (Single frame from Movie 4).

    Article Snippet: Sucrosome formation was induced by incubation of cells grown in AMEM (Life Technologies, Inc.) medium, supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 IU/mL of penicillin, 100 µg/mL streptomycin, and 100 µg/mL G418 containing 60 mM sucrose in for 24 h. Confocal Microscopy—Laser scanning confocal microscopy to monitor GFP, filipin and Alexa 568 and Alexa 594 fluorescence, was performed using a Zeiss 510 LSCM.

    Techniques: Labeling, Marker, Staining, Expressing, Confocal Microscopy

    The content and cellular distributions of ergosterol are abnormal under ypkA loss of function. (A) 1 × 10 3 conidia of wild-type strain and a cell amount of Δ ypkA mutant were inocul ated in liquid YG culture and incubated at 37°C for 120 h before ergosterol extraction and quantification by HPLC (see section “Materials and Methods” for details). (B) 1 × 10 7 conidia of both wild-type and niiA::ypkA strains were inoculated in liquid AMM supplemented with MN or AT and incubated at 37°C for 24 h before ergosterol extraction and quantification by HPLC. Experiments were performed in triplicate and the results are the mean ± SD. Results show the ergosterol concentration normalized by the mycelia dry weight. ∗ Statistically significant (Student’s t -test; p ≤ 0.05). (C) 1 × 10 5 conidia of niiA::ypkA strain were inoculated in liquid AMM supplemented with MN or AT and incubated at 37°C for 12 h. The germilings were stained with 25 μg/ml of filipin for 5 min, observed in a fluorescence microscope and photographed. Dashed bars = 20 μm. Solid bars = 10 μm.

    Journal: Frontiers in Microbiology

    Article Title: The AGC Kinase YpkA Regulates Sphingolipids Biosynthesis and Physically Interacts With SakA MAP Kinase in Aspergillus fumigatus

    doi: 10.3389/fmicb.2018.03347

    Figure Lengend Snippet: The content and cellular distributions of ergosterol are abnormal under ypkA loss of function. (A) 1 × 10 3 conidia of wild-type strain and a cell amount of Δ ypkA mutant were inocul ated in liquid YG culture and incubated at 37°C for 120 h before ergosterol extraction and quantification by HPLC (see section “Materials and Methods” for details). (B) 1 × 10 7 conidia of both wild-type and niiA::ypkA strains were inoculated in liquid AMM supplemented with MN or AT and incubated at 37°C for 24 h before ergosterol extraction and quantification by HPLC. Experiments were performed in triplicate and the results are the mean ± SD. Results show the ergosterol concentration normalized by the mycelia dry weight. ∗ Statistically significant (Student’s t -test; p ≤ 0.05). (C) 1 × 10 5 conidia of niiA::ypkA strain were inoculated in liquid AMM supplemented with MN or AT and incubated at 37°C for 12 h. The germilings were stained with 25 μg/ml of filipin for 5 min, observed in a fluorescence microscope and photographed. Dashed bars = 20 μm. Solid bars = 10 μm.

    Article Snippet: Filipin and GFP were visualized using 49 and 38 HE filter sets (Carl Zeiss), respectively, and 100× magnification oil immersion objective (Plan-Apochromat; NA 1.4).

    Techniques: Mutagenesis, Incubation, High Performance Liquid Chromatography, Concentration Assay, Staining, Fluorescence, Microscopy

    Characterization of stem cell populations obtained from skin biopsies or already established skin fibroblast cultures. ( A - D ) Phase contrast images of hSKIN-MASC at the third passage in culture: A - B ) hSKIN-MASC obtained from skin biopsies of a healthy donor ( A ) and a NPC patient ( B ). C - D ) hSKIN-MASC obtained from already established skin fibroblasts from healthy donor ( C ) and NPC patient ( D ). ( E ) Surface immunophenotype: representative flow cytometry histograms of skin derived stem cell cultures. Plots show isotype control IgG-staining profile (green histograms) versus specific antibody staining (red histograms). ( F - H ) Pluripotent state specific transcription factor expression: representative fluorescence images of Oct-4 (green fluorescence; F ), Nanog (green fluorescence; G ) and Sox-2 expression (green fluorescence; H ) localized in the nuclei of skin derived stem cell cultures. Nuclei are depicted by the blue fluorescence of DAPI staining ( F-H ). I) Quantification of the percentage of cells expressing Oct4 (white bars), Nanog (black bars) and Sox-2 (gray bars) in hSKIN-MASC obtained both from healthy donors (CTRL, n = 3) and NPC (n = 3) skin biopsies or healthy donors (CTRL, n = 3) and NPC (n = 3) skin fibroblast cultures. At least 400 cells have been counted for each cell line. Data are presented as mean ± SD of 3 independent experiments. ( J - K ) Nestin expression (red fluorescence) in hSKIN-MASC obtained from a healthy donor ( J ) and a NPC patient ( K ). ( L - N ) Unesterified cholesterol accumulation: images obtained after performing the filipin staining (blue fluorescence) in hSKIN-MASC derived from a healthy donor skin biopsy ( L ), a NPC patient skin biopsy ( M) and a NPC skin fibroblast cell line ( N ) , respectively.

    Journal: Orphanet Journal of Rare Diseases

    Article Title: A human neuronal model of Niemann Pick C disease developed from stem cells isolated from patient's skin

    doi: 10.1186/1750-1172-8-34

    Figure Lengend Snippet: Characterization of stem cell populations obtained from skin biopsies or already established skin fibroblast cultures. ( A - D ) Phase contrast images of hSKIN-MASC at the third passage in culture: A - B ) hSKIN-MASC obtained from skin biopsies of a healthy donor ( A ) and a NPC patient ( B ). C - D ) hSKIN-MASC obtained from already established skin fibroblasts from healthy donor ( C ) and NPC patient ( D ). ( E ) Surface immunophenotype: representative flow cytometry histograms of skin derived stem cell cultures. Plots show isotype control IgG-staining profile (green histograms) versus specific antibody staining (red histograms). ( F - H ) Pluripotent state specific transcription factor expression: representative fluorescence images of Oct-4 (green fluorescence; F ), Nanog (green fluorescence; G ) and Sox-2 expression (green fluorescence; H ) localized in the nuclei of skin derived stem cell cultures. Nuclei are depicted by the blue fluorescence of DAPI staining ( F-H ). I) Quantification of the percentage of cells expressing Oct4 (white bars), Nanog (black bars) and Sox-2 (gray bars) in hSKIN-MASC obtained both from healthy donors (CTRL, n = 3) and NPC (n = 3) skin biopsies or healthy donors (CTRL, n = 3) and NPC (n = 3) skin fibroblast cultures. At least 400 cells have been counted for each cell line. Data are presented as mean ± SD of 3 independent experiments. ( J - K ) Nestin expression (red fluorescence) in hSKIN-MASC obtained from a healthy donor ( J ) and a NPC patient ( K ). ( L - N ) Unesterified cholesterol accumulation: images obtained after performing the filipin staining (blue fluorescence) in hSKIN-MASC derived from a healthy donor skin biopsy ( L ), a NPC patient skin biopsy ( M) and a NPC skin fibroblast cell line ( N ) , respectively.

    Article Snippet: After washing them with PBS, the cells were incubated with 1.5 mg of glycine/ml PBS for 10 minutes, stained with filipin (0.05 mg/ml, in PBS 10% FCS) for 2 hours and examined using a Zeiss fluorescence microscope.

    Techniques: Flow Cytometry, Cytometry, Derivative Assay, Staining, Expressing, Fluorescence

    Fibroblast cell culture from the conjunctival biopsy of the proband. A : Fibroblastic cellular outgrowth from the conjunctival biopsy tissue. B : Following one week of cell culture, cellular confluence of fibroblasts is achieved. C : Following the addition of filipin stain, there is no evidence of cholesterol in the cultured conjunctival fibroblasts. D : Cells cultured with the addition of low density lipoprotein (LDL), acted as a positive control showing evidence of cholesterol with filipin staining. (Bar=100 microns).

    Journal: Molecular Vision

    Article Title: Analysis of conjunctival fibroblasts from a proband with Schnyder corneal dystrophy

    doi:

    Figure Lengend Snippet: Fibroblast cell culture from the conjunctival biopsy of the proband. A : Fibroblastic cellular outgrowth from the conjunctival biopsy tissue. B : Following one week of cell culture, cellular confluence of fibroblasts is achieved. C : Following the addition of filipin stain, there is no evidence of cholesterol in the cultured conjunctival fibroblasts. D : Cells cultured with the addition of low density lipoprotein (LDL), acted as a positive control showing evidence of cholesterol with filipin staining. (Bar=100 microns).

    Article Snippet: The filipin dye was excited with epi-illumination using ultraviolet light (Axioplan, Zeiss Light Microscope, Carl Zeiss), and fluorescence was viewed through a 510 nm barrier filter.

    Techniques: Cell Culture, Staining, Positive Control