hs27 hdfs (ATCC)

ATCC hs27 hdfs
$PTS1(-SKL)-protein import in early-, middle-, and late-passage <t>HDFs.</t> (A) Semi-intact IMR90 HDFs were incubated at 37°C with biotinylated luciferase in an in vitro import reaction. After 45 min, the cells were centrifuged and homogenized, and an organelle pellet/peroxisome fraction was prepared. The level of import in equivalent portions of the organelle pellets was determined by ELISA. Values presented (means and ranges of duplicate samples) are absorbance units (at 490 nm) with time zero values subtracted. E, Early-passage cells; M, middle-passage cells; L, late-passage cells. (B) Semi-intact <t>Hs27</t> HDFs were incubated with biotinylated luciferase as in (A) except that import was assayed directly in cells. (C) Streptolysin-O–permeabilized IMR90 cells were incubated with luciferase for 1 h at 37°C. The cells were then fixed, treated with detergent, and examined by indirect immunofluorescence with anti-luciferase antibodies and CY3-labeled secondary antibodies. Bottom, phase-contrast images of the cells at top. The graph represents quantification of the import results. The number of detectable peroxisomes was determined by counting immunoreactive structures in each of the cells. For this analysis, a greater number of cells were evaluated than are shown in the immunofluorescence image. Values presented are the means ± SD.$
Hs27 Hdfs, supplied by ATCC, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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imr90 (Coriell Institute for Medical Research)

Coriell Institute for Medical Research imr90
$PTS1(-SKL)-protein import in early-, middle-, and late-passage <t>HDFs.</t> (A) Semi-intact IMR90 HDFs were incubated at 37°C with biotinylated luciferase in an in vitro import reaction. After 45 min, the cells were centrifuged and homogenized, and an organelle pellet/peroxisome fraction was prepared. The level of import in equivalent portions of the organelle pellets was determined by ELISA. Values presented (means and ranges of duplicate samples) are absorbance units (at 490 nm) with time zero values subtracted. E, Early-passage cells; M, middle-passage cells; L, late-passage cells. (B) Semi-intact <t>Hs27</t> HDFs were incubated with biotinylated luciferase as in (A) except that import was assayed directly in cells. (C) Streptolysin-O–permeabilized IMR90 cells were incubated with luciferase for 1 h at 37°C. The cells were then fixed, treated with detergent, and examined by indirect immunofluorescence with anti-luciferase antibodies and CY3-labeled secondary antibodies. Bottom, phase-contrast images of the cells at top. The graph represents quantification of the import results. The number of detectable peroxisomes was determined by counting immunoreactive structures in each of the cells. For this analysis, a greater number of cells were evaluated than are shown in the immunofluorescence image. Values presented are the means ± SD.$
Imr90, supplied by Coriell Institute for Medical Research, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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$PTS1(-SKL)-protein import in early-, middle-, and late-passage HDFs. (A) Semi-intact IMR90 HDFs were incubated at 37°C with biotinylated luciferase in an in vitro import reaction. After 45 min, the cells were centrifuged and homogenized, and an organelle pellet/peroxisome fraction was prepared. The level of import in equivalent portions of the organelle pellets was determined by ELISA. Values presented (means and ranges of duplicate samples) are absorbance units (at 490 nm) with time zero values subtracted. E, Early-passage cells; M, middle-passage cells; L, late-passage cells. (B) Semi-intact Hs27 HDFs were incubated with biotinylated luciferase as in (A) except that import was assayed directly in cells. (C) Streptolysin-O–permeabilized IMR90 cells were incubated with luciferase for 1 h at 37°C. The cells were then fixed, treated with detergent, and examined by indirect immunofluorescence with anti-luciferase antibodies and CY3-labeled secondary antibodies. Bottom, phase-contrast images of the cells at top. The graph represents quantification of the import results. The number of detectable peroxisomes was determined by counting immunoreactive structures in each of the cells. For this analysis, a greater number of cells were evaluated than are shown in the immunofluorescence image. Values presented are the means ± SD.$

Journal:

Article Title: Peroxisome Senescence in Human Fibroblasts

doi: 10.1091/mbc.E02-06-0322

Figure Lengend Snippet: PTS1(-SKL)-protein import in early-, middle-, and late-passage HDFs. (A) Semi-intact IMR90 HDFs were incubated at 37°C with biotinylated luciferase in an in vitro import reaction. After 45 min, the cells were centrifuged and homogenized, and an organelle pellet/peroxisome fraction was prepared. The level of import in equivalent portions of the organelle pellets was determined by ELISA. Values presented (means and ranges of duplicate samples) are absorbance units (at 490 nm) with time zero values subtracted. E, Early-passage cells; M, middle-passage cells; L, late-passage cells. (B) Semi-intact Hs27 HDFs were incubated with biotinylated luciferase as in (A) except that import was assayed directly in cells. (C) Streptolysin-O–permeabilized IMR90 cells were incubated with luciferase for 1 h at 37°C. The cells were then fixed, treated with detergent, and examined by indirect immunofluorescence with anti-luciferase antibodies and CY3-labeled secondary antibodies. Bottom, phase-contrast images of the cells at top. The graph represents quantification of the import results. The number of detectable peroxisomes was determined by counting immunoreactive structures in each of the cells. For this analysis, a greater number of cells were evaluated than are shown in the immunofluorescence image. Values presented are the means ± SD.

Article Snippet: Early-passage IMR90 and Hs27 HDFs, obtained from the National Institute of Aging, Aging Cell Repository/Coriell Institute for Medical Research (Camden, NJ), and ATCC (Manassas, VA), respectively, were cultured in DMEM supplemented with 10% fetal bovine serum (Life Technologies, Grand Island, NY), penicillin, and streptomycin.

Techniques: Incubation, Luciferase, In Vitro, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Labeling

Peroxisomes in early-, middle-, and late-passage HDFs. (A) The peroxisomal membrane marker PMP70 was examined by indirect immunofluorescence in early (E)-, middle (M)-, and late (L)-passage HDFs. (B and C) PMP70, serine-lysine-leucine (SKL)-containing proteins, and catalase were examined by indirect immunofluorescence in cocultured early- (E) and late- (L) passage IMR90 (B) or Hs27 (C) HDFs.

Journal:

Article Title: Peroxisome Senescence in Human Fibroblasts

doi: 10.1091/mbc.E02-06-0322

Figure Lengend Snippet: Peroxisomes in early-, middle-, and late-passage HDFs. (A) The peroxisomal membrane marker PMP70 was examined by indirect immunofluorescence in early (E)-, middle (M)-, and late (L)-passage HDFs. (B and C) PMP70, serine-lysine-leucine (SKL)-containing proteins, and catalase were examined by indirect immunofluorescence in cocultured early- (E) and late- (L) passage IMR90 (B) or Hs27 (C) HDFs.

Techniques: Membrane, Marker, Immunofluorescence

Catalase latency. IMR90 HDFs were treated with increasing concentrations of digitonin, and the levels of catalase (▪) and lactate dehydrogenase (●) were determined. Data are presented as percentage of total cellular activity (set at 100), which was determined in the presence of 1% Triton X-100. Solid symbols, early-passage cells; open symbols, late-passage cells.

Journal:

Article Title: Peroxisome Senescence in Human Fibroblasts

doi: 10.1091/mbc.E02-06-0322

Figure Lengend Snippet: Catalase latency. IMR90 HDFs were treated with increasing concentrations of digitonin, and the levels of catalase (▪) and lactate dehydrogenase (●) were determined. Data are presented as percentage of total cellular activity (set at 100), which was determined in the presence of 1% Triton X-100. Solid symbols, early-passage cells; open symbols, late-passage cells.

Techniques: Activity Assay

Peroxisomal import of PTS1(−SKL and −KANL)-tagged constructs in early- and late-passage HDFs. (A) Early (E)- and late (L)-passage Hs27 HDFs were nuclear-microinjected with a plasmid encoding GFP-SKL or GFP-KANL as indicated. Live images were taken 18 or 45 h after injection on an inverted microscope. (B) Late-passage Hs27 HDFs were nuclear-microinjected with plasmids encoding DsRed2-SKL and GFP-KANL, incubated for 42 h, fixed, and imaged.

Journal:

Article Title: Peroxisome Senescence in Human Fibroblasts

doi: 10.1091/mbc.E02-06-0322

Figure Lengend Snippet: Peroxisomal import of PTS1(−SKL and −KANL)-tagged constructs in early- and late-passage HDFs. (A) Early (E)- and late (L)-passage Hs27 HDFs were nuclear-microinjected with a plasmid encoding GFP-SKL or GFP-KANL as indicated. Live images were taken 18 or 45 h after injection on an inverted microscope. (B) Late-passage Hs27 HDFs were nuclear-microinjected with plasmids encoding DsRed2-SKL and GFP-KANL, incubated for 42 h, fixed, and imaged.

Techniques: Construct, Plasmid Preparation, Injection, Inverted Microscopy, Incubation

Association of Pex5p with organelle membranes. (A) Pex5p accumulates on organelles of aging HDFs. Pex5p was immunoprecipitated from detergent-solubilized organelles of early-passage (E), middle-passage (M), or late-passage (L) IMR90 HDFs using anti-Pex5p antisera (α5) or preimmune sera (PI), as indicated, and immunoblotted with anti-Pex5p antisera. Organellar PMP70 was also examined by immunoblotting. The graph represents quantification of the α5 immunoblot with a Fujifilm LAS100plus luminescent image analyzer. The units on the ordinate are arbitrary. Equivalent results were obtained in three different experiments. (B) Pex5p accumulates on the outside of organelles from late-passage cells. Organelles from late-passage IMR90 HDFs were treated with proteinase K and Triton X-100 or untreated, as indicated, and the resultant organelles were immunoblotted with anti-Pex5p or anti-catalase antisera.

Journal:

Article Title: Peroxisome Senescence in Human Fibroblasts

doi: 10.1091/mbc.E02-06-0322

Figure Lengend Snippet: Association of Pex5p with organelle membranes. (A) Pex5p accumulates on organelles of aging HDFs. Pex5p was immunoprecipitated from detergent-solubilized organelles of early-passage (E), middle-passage (M), or late-passage (L) IMR90 HDFs using anti-Pex5p antisera (α5) or preimmune sera (PI), as indicated, and immunoblotted with anti-Pex5p antisera. Organellar PMP70 was also examined by immunoblotting. The graph represents quantification of the α5 immunoblot with a Fujifilm LAS100plus luminescent image analyzer. The units on the ordinate are arbitrary. Equivalent results were obtained in three different experiments. (B) Pex5p accumulates on the outside of organelles from late-passage cells. Organelles from late-passage IMR90 HDFs were treated with proteinase K and Triton X-100 or untreated, as indicated, and the resultant organelles were immunoblotted with anti-Pex5p or anti-catalase antisera.

Techniques: Immunoprecipitation, Western Blot

Hydrogen peroxide accumulates in aging HDFs and inhibits peroxisomal protein import. (A) IMR90 HDFs at early (E), middle (M), and late (L) passage were examined for the generation of hydrogen peroxide using the fluorescent dye 2′,7′-dichlorofluorescin diacetate. (B) Hydrogen peroxide in cocultured early- and late-passage Hs27 HDFs. Early-passage cells are readily identified as those having endocytosed red FluoSphere microspheres. (C) Effect of hydrogen peroxide on PTS1 (−SKL)-protein import. Early-passage IMR90 HDFs were pretreated with hydrogen peroxide or untreated as indicated, and peroxisomal protein import was examined as in Figure Figure1A.1A. Results presented (mean ± one SD) are pooled from five experiments and normalized to the untreated control cell value (arbitrarily set at 100) to permit comparison. (D) Effect of hydrogen peroxide on localization of the PTS1 receptor. Pex5p was examined by indirect immunofluorescence in early-passage HDFs pretreated with hydrogen peroxide or untreated as indicated.

Journal:

Article Title: Peroxisome Senescence in Human Fibroblasts

doi: 10.1091/mbc.E02-06-0322

Figure Lengend Snippet: Hydrogen peroxide accumulates in aging HDFs and inhibits peroxisomal protein import. (A) IMR90 HDFs at early (E), middle (M), and late (L) passage were examined for the generation of hydrogen peroxide using the fluorescent dye 2′,7′-dichlorofluorescin diacetate. (B) Hydrogen peroxide in cocultured early- and late-passage Hs27 HDFs. Early-passage cells are readily identified as those having endocytosed red FluoSphere microspheres. (C) Effect of hydrogen peroxide on PTS1 (−SKL)-protein import. Early-passage IMR90 HDFs were pretreated with hydrogen peroxide or untreated as indicated, and peroxisomal protein import was examined as in Figure Figure1A.1A. Results presented (mean ± one SD) are pooled from five experiments and normalized to the untreated control cell value (arbitrarily set at 100) to permit comparison. (D) Effect of hydrogen peroxide on localization of the PTS1 receptor. Pex5p was examined by indirect immunofluorescence in early-passage HDFs pretreated with hydrogen peroxide or untreated as indicated.

Techniques: Control, Comparison, Immunofluorescence

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