Aggresomes: A Cellular Response to Misfolded Proteins methods
Show snapshot details
On this page
This experiment, in seven questions
Jump straight to the part of the recipe you need. Data and provenance labels stay close to the action they support.
Shopping and prep list
What do I need before I start?
Biological model pending
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Before you run
What should be confirmed before execution?
First confirmation
Species or subject information is missing.
Confirm before execution
Protocol does not list structured equipment.
Confirm before execution
This page shows passages quoted from the paper. Equipment, materials, times, and temperatures are hidden until the page is rebuilt from the full paper and checked by a reviewer. Check the paper before using this method.
Confirm before execution
Open the source paper before finalizing run-specific details.
Procurement checkpoint
Use source-stated vendors where present. Treat mapped products as sourcing options unless the page marks an exact source match.
Open quote workflowStep-by-step procedure
What do I do, in order?
Cell Lines and Cell Culture
Human embryonic kidney 293 (HEK) cells were maintained in DME and transfected as described previously ( ). Stable GFP-CFTR-expressing cell lines were maintained in the same media with the addition of 2 mg/ml G418. Transient transfections were carried out by adding plasmid DNA as a calcium phosphate precipitate ( ). Stable cell lines were prepared by transfection with Lipofectamine ( GIBCO BRL, Gaithersburg, MD) followed after 48 h by selection of transformed cells by growth in 1 mg/ml Geneticin (G418). These G418 resistant cells were then analyzed by flow cytometry and the top 20% fluorescent cells were isolated. This population of cells was then expanded and assayed for expression by immunoblotting with antibodies to GFP and CFTR.
Pulse-Chase Experiments and Immunoblotting
Cells were pulse-labeled with [ 35 S]methionine/cysteine for 15 min, harvested, and immunoprecipitated as described previously ( ). Loading volumes were normalized to the sample with the lowest TCA precipitable cpm. For immunoblotting, cell pellets from washed and transfected HEK cells were lysed in 250 µl of ice-cold IPB buffer (10 mM Tris-HCl, pH 7.5, 5 mM EDTA, 1% NP-40, 0.5% deoxycholate, and 150 mM NaCl) plus protease inhibitors (100 µM TLCK, 100 µM TPCK, and 1 mM PMSF) for 30 min on ice. Insoluble material was recovered by centrifugation at 13,000 g for 15 min and solubilized in 50 µl 10 mM Tris-HCl, 1% SDS for 10 min at room temperature. After addition of 200 µl IPB, samples were sonicated for 20 s with a tip sonicator. Endoglycosidase H ( Boehringer Mannheim, Mannheim, Germany) digestion was performed on immunoprecipitates overnight at 37°C in th...
Fluorescence Microscopy
Cells were seeded onto No. 1 coverslips. For drug treatments, ALLN ( Calbiochem, La Jolla, CA) 5-10 µg/ml and nocodozole ( Sigma Chemical Co. ) 10 µg/ml in DMSO were added to the culture medium 12 h before fixation. Cells were fixed in -20°C methanol (6 min), 50% methanol/50% acetone (6 min at room temperature), 4% paraformaldehyde (followed by a 0.5% Triton X-100 permeabilization for 15 min), with no observable differences in aggresome morphology, or relative position to markers described in the results. After fixation, cells were washed extensively in PBS and blocked with 10% BSA for 10 min. Primary antibody was incubated for 30 min to 12 h. Cells were washed 5 × 5 min in PBS and incubated with fluorophore conjugated secondary antibodies at a 1:100 dilution of 1 mg/ml stock. Cells were washed again (10 × 2 min each) and then incubated for 3 min...
EM
Cells were grown to 85% confluence in 100-mm dishes and treated overnight with 10 µg/ml ALLN, washed once in PBS, and then fixed in freshly made AMT buffer (100 mM Hepes, pH 7.4, 5 mM MgCl 2, 1% glycerol, and 2% glutaraldehyde [EMS]) for 35 min at room temperature. Cells were washed twice in phosphate buffer (100 mM phosphate buffer, pH 7.5), scraped, and collected by centrifugation for 10 min at 10,000 g. The fixed pellet of cells was then postfixed for 45 min in 2% OsO 4, and then washed extensively in water. En bloc staining in 1% UA for 1 h was followed by a series of graded ethanol dehydrations (25, 50, 75, 85, 95, and 100%) followed by overnight incubation in 1:1 100% ethanol/LR White medium grade resin, or Polybed resin (Polysciences, Inc., Warrington, PA). After two changes of fresh 100% resin, the cell pellets were transferred to gelatin capsules and polymerized in fr...
Preparation of Aggresomes
Because the aggresome consists of a juxtanuclear cap of vimentin, the technique of Starger ( ) was modified to optimize coisolation of GFP-CFTR and IF. In brief, cells were grown to 85% confluency in 100-mm dishes and treated with 10 µg/ml ALLN for 12 h before isolation. Cells were washed twice in PBSa (6 mM sodium-potassium phosphate buffer, 170 mM NaCl, 3 mM KCl), scraped, and collected for 3 min at 2,500 g. Washed cells were resuspended in 1 ml PBSa per 100-mm plate and passaged through a 25-gauge needle three to four times until bright-field microscopy revealed the majority of cells were disrupted. This material was washed by resuspension and sedimentation at 2,000 g three times in PBSa. The resulting material was examined by fluorescence microscopy for the presence of GFP-containing isolated aggresomes. This resulting cellular fraction enriched for aggresomes was collected...
Measurement outputs
What raw and processed outputs should exist?
Gait recovery and locomotor function
- Raw artifact
- Per-run gait capture with paw placement, timing, and stride features for each animal
- Processed artifact
- Cleaned gait metrics table and recovery trend summary across timepoints
- Reported as
- Group comparisons of gait indices, stride metrics, or recovery curves
Microscopy-based tissue or cell-state imaging
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
Analysis plan
How should the outputs become interpretable results?
Acquisition
Collect raw experimental outputs with enough metadata to preserve sample identity, condition, and timing.
inferred from protocolPreprocessing / cleaning
Review raw outputs for quality, remove unusable captures, and organize the data into a comparison-ready table or image set.
inferred from protocolScoring or quantification
Quantify the primary readouts for this experiment: Gait recovery and locomotor function; Microscopy-based tissue or cell-state imaging.
from paperStatistical comparison
Statistical method not yet structured for this page.
inferred from protocolReporting output
Report representative outputs alongside summary comparisons for Gait recovery and locomotor function, Microscopy-based tissue or cell-state imaging.
inferred from protocolSource and audit
What supports the facts on this page?
Evidence quotes (5)
Human embryonic kidney 293 (HEK) cells were maintained in DME and transfected as described previously ( ). Stable GFP-CFTR-expressing cell lines were maintained in the same media with the addition of 2 mg/ml G418. Transient transfections were carried out by adding plasmid DNA as a calcium phosphate precipitate ( ). Stable cell lines were prepared by transfection with Lipofectamine ( GIBCO BRL, Gaithersburg, MD) followed after 48 h by selection of transformed cells by growth in 1 mg/ml Geneticin (G418). These G418 resistant cells were then analyzed by flow cytometry and the top 20% fluorescent cells were isolated. This population of cells was then expanded and assayed for expression by immunoblotting with antibodies to GFP and CFTR.
Cells were pulse-labeled with [ 35 S]methionine/cysteine for 15 min, harvested, and immunoprecipitated as described previously ( ). Loading volumes were normalized to the sample with the lowest TCA precipitable cpm. For immunoblotting, cell pellets from washed and transfected HEK cells were lysed in 250 µl of ice-cold IPB buffer (10 mM Tris-HCl, pH 7.5, 5 mM EDTA, 1% NP-40, 0.5% deoxycholate, and 150 mM NaCl) plus protease inhibitors (100 µM TLCK, 100 µM TPCK, and 1 mM PMSF) for 30 min on ice. Insoluble material was recovered by centrifugation at 13,000 g for 15 min and solubilized in 50 µl 10 mM Tris-HCl, 1% SDS for 10 min at room temperature. After addition of 200 µl IPB, samples were sonicated for 20 s with a tip sonicator. Endoglycosidase H ( Boehringer Mannheim, Mannheim, Germany) digestion was performed on immunoprecipitates overnight at 37°C in the buffer supplied by the manufacturer. Gel autoradiograms were quantified as described previously ( ). For immunoblotting, cell fractions normalized for total protein were separated on 7% SDS-PAGE and electroblotted. Chemiluminescent detection was carried out with the Renaissance detection kit ( New...
Cells were seeded onto No. 1 coverslips. For drug treatments, ALLN ( Calbiochem, La Jolla, CA) 5-10 µg/ml and nocodozole ( Sigma Chemical Co. ) 10 µg/ml in DMSO were added to the culture medium 12 h before fixation. Cells were fixed in -20°C methanol (6 min), 50% methanol/50% acetone (6 min at room temperature), 4% paraformaldehyde (followed by a 0.5% Triton X-100 permeabilization for 15 min), with no observable differences in aggresome morphology, or relative position to markers described in the results. After fixation, cells were washed extensively in PBS and blocked with 10% BSA for 10 min. Primary antibody was incubated for 30 min to 12 h. Cells were washed 5 × 5 min in PBS and incubated with fluorophore conjugated secondary antibodies at a 1:100 dilution of 1 mg/ml stock. Cells were washed again (10 × 2 min each) and then incubated for 3 min in PBS + bisbenzamide ( Sigma Chemical Co. ) at 10 µg/ml to stain the DNA. Cells were washed a final time, mounted onto slides in 50% PBS/ 50% glycerol and viewed at 40 × /1.25 NA or 63 × /1.4 NA with a Zeiss Axiophot microscope. Images were obtained with Metamorph software ( Universal Im...
Cells were grown to 85% confluence in 100-mm dishes and treated overnight with 10 µg/ml ALLN, washed once in PBS, and then fixed in freshly made AMT buffer (100 mM Hepes, pH 7.4, 5 mM MgCl 2, 1% glycerol, and 2% glutaraldehyde [EMS]) for 35 min at room temperature. Cells were washed twice in phosphate buffer (100 mM phosphate buffer, pH 7.5), scraped, and collected by centrifugation for 10 min at 10,000 g. The fixed pellet of cells was then postfixed for 45 min in 2% OsO 4, and then washed extensively in water. En bloc staining in 1% UA for 1 h was followed by a series of graded ethanol dehydrations (25, 50, 75, 85, 95, and 100%) followed by overnight incubation in 1:1 100% ethanol/LR White medium grade resin, or Polybed resin (Polysciences, Inc., Warrington, PA). After two changes of fresh 100% resin, the cell pellets were transferred to gelatin capsules and polymerized in fresh resin overnight at 45-60°C. Gold sections were cut and collected onto copper grids and stained with 1% UA for 3 min followed by 5 min in lead citrate to generate final contrast, before viewing in a JOEL electron microscope. For immunogold EM experiments, isolated aggresomes (see below...
Because the aggresome consists of a juxtanuclear cap of vimentin, the technique of Starger ( ) was modified to optimize coisolation of GFP-CFTR and IF. In brief, cells were grown to 85% confluency in 100-mm dishes and treated with 10 µg/ml ALLN for 12 h before isolation. Cells were washed twice in PBSa (6 mM sodium-potassium phosphate buffer, 170 mM NaCl, 3 mM KCl), scraped, and collected for 3 min at 2,500 g. Washed cells were resuspended in 1 ml PBSa per 100-mm plate and passaged through a 25-gauge needle three to four times until bright-field microscopy revealed the majority of cells were disrupted. This material was washed by resuspension and sedimentation at 2,000 g three times in PBSa. The resulting material was examined by fluorescence microscopy for the presence of GFP-containing isolated aggresomes. This resulting cellular fraction enriched for aggresomes was collected a final time at 2,000 g and resuspended in 200 µl of PBS/1% BSA. This served as the starting material for the immunoelectron microscopy techniques described above.
Machine-readable layer
[
{
"@context": "https://schema.org",
"@type": "HowTo",
"name": "Aggresomes: A Cellular Response to Misfolded Proteins methods",
"description": "Protocol for Aggresomes: A Cellular Response to Misfolded Proteins methods",
"step": [
{
"@type": "HowToStep",
"position": 1,
"name": "Cell Lines and Cell Culture",
"text": "Human embryonic kidney 293 (HEK) cells were maintained in DME and transfected as described previously ( ). Stable GFP-CFTR-expressing cell lines were maintained in the same media with the addition of 2 mg/ml G418. Transient transfections were carried out by adding plasmid DNA as a calcium phosphate precipitate ( ). Stable cell lines were prepared by transfection with Lipofectamine ( GIBCO BRL, Gaithersburg, MD) followed after 48 h by selection of transformed cells by growth in 1 mg/ml Geneticin (G418). These G418 resistant cells were then analyzed by flow cytometry and the top 20% fluorescent cells were isolated. This population of cells was then expanded and assayed for expression by immunoblotting with antibodies to GFP and CFTR."
},
{
"@type": "HowToStep",
"position": 2,
"name": "Pulse-Chase Experiments and Immunoblotting",
"text": "Cells were pulse-labeled with [ 35 S]methionine/cysteine for 15 min, harvested, and immunoprecipitated as described previously ( ). Loading volumes were normalized to the sample with the lowest TCA precipitable cpm. For immunoblotting, cell pellets from washed and transfected HEK cells were lysed in 250 µl of ice-cold IPB buffer (10 mM Tris-HCl, pH 7.5, 5 mM EDTA, 1% NP-40, 0.5% deoxycholate, and 150 mM NaCl) plus protease inhibitors (100 µM TLCK, 100 µM TPCK, and 1 mM PMSF) for 30 min on ice. Insoluble material was recovered by centrifugation at 13,000 g for 15 min and solubilized in 50 µl 10 mM Tris-HCl, 1% SDS for 10 min at room temperature. After addition of 200 µl IPB, samples were sonicated for 20 s with a tip sonicator. Endoglycosidase H ( Boehringer Mannheim, Mannheim, Germany) digestion was performed on immunoprecipitates overnight at 37°C in th..."
},
{
"@type": "HowToStep",
"position": 3,
"name": "Fluorescence Microscopy",
"text": "Cells were seeded onto No. 1 coverslips. For drug treatments, ALLN ( Calbiochem, La Jolla, CA) 5-10 µg/ml and nocodozole ( Sigma Chemical Co. ) 10 µg/ml in DMSO were added to the culture medium 12 h before fixation. Cells were fixed in -20°C methanol (6 min), 50% methanol/50% acetone (6 min at room temperature), 4% paraformaldehyde (followed by a 0.5% Triton X-100 permeabilization for 15 min), with no observable differences in aggresome morphology, or relative position to markers described in the results. After fixation, cells were washed extensively in PBS and blocked with 10% BSA for 10 min. Primary antibody was incubated for 30 min to 12 h. Cells were washed 5 × 5 min in PBS and incubated with fluorophore conjugated secondary antibodies at a 1:100 dilution of 1 mg/ml stock. Cells were washed again (10 × 2 min each) and then incubated for 3 min..."
},
{
"@type": "HowToStep",
"position": 4,
"name": "EM",
"text": "Cells were grown to 85% confluence in 100-mm dishes and treated overnight with 10 µg/ml ALLN, washed once in PBS, and then fixed in freshly made AMT buffer (100 mM Hepes, pH 7.4, 5 mM MgCl 2, 1% glycerol, and 2% glutaraldehyde [EMS]) for 35 min at room temperature. Cells were washed twice in phosphate buffer (100 mM phosphate buffer, pH 7.5), scraped, and collected by centrifugation for 10 min at 10,000 g. The fixed pellet of cells was then postfixed for 45 min in 2% OsO 4, and then washed extensively in water. En bloc staining in 1% UA for 1 h was followed by a series of graded ethanol dehydrations (25, 50, 75, 85, 95, and 100%) followed by overnight incubation in 1:1 100% ethanol/LR White medium grade resin, or Polybed resin (Polysciences, Inc., Warrington, PA). After two changes of fresh 100% resin, the cell pellets were transferred to gelatin capsules and polymerized in fr..."
},
{
"@type": "HowToStep",
"position": 5,
"name": "Preparation of Aggresomes",
"text": "Because the aggresome consists of a juxtanuclear cap of vimentin, the technique of Starger ( ) was modified to optimize coisolation of GFP-CFTR and IF. In brief, cells were grown to 85% confluency in 100-mm dishes and treated with 10 µg/ml ALLN for 12 h before isolation. Cells were washed twice in PBSa (6 mM sodium-potassium phosphate buffer, 170 mM NaCl, 3 mM KCl), scraped, and collected for 3 min at 2,500 g. Washed cells were resuspended in 1 ml PBSa per 100-mm plate and passaged through a 25-gauge needle three to four times until bright-field microscopy revealed the majority of cells were disrupted. This material was washed by resuspension and sedimentation at 2,000 g three times in PBSa. The resulting material was examined by fluorescence microscopy for the presence of GFP-containing isolated aggresomes. This resulting cellular fraction enriched for aggresomes was collected..."
}
],
"tool": [],
"supply": [],
"isBasedOn": {
"@type": "ScholarlyArticle",
"headline": "Aggresomes: A Cellular Response to Misfolded Proteins",
"datePublished": "1998",
"author": [
{
"@type": "Person",
"name": "Jennifer A. Johnston"
},
{
"@type": "Person",
"name": "Cristina L. Ward"
},
{
"@type": "Person",
"name": "Ron R. Kopito"
}
],
"identifier": "10.1083/jcb.143.7.1883"
}
},
{
"@context": "https://schema.org",
"@type": "BreadcrumbList",
"itemListElement": [
{
"@type": "ListItem",
"position": 1,
"name": "Experiments",
"item": "https://replicatescience.com/experiments"
},
{
"@type": "ListItem",
"position": 2,
"name": "Aggresomes: A Cellular Response to Misfolded Proteins methods",
"item": "https://replicatescience.com/experiments/aggresomes-a-cellular-response-to-misfolded-proteins-methods-jennifer-a-johnston-pmc2175217/aggresomes-a-cellular-response-to-misfolded-proteins-mlpgvlua"
}
]
}
]