Cell-intrinsic lysosomal lipolysis is essential for macrophage alternative activation 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?
Methods
C57BL/6J mice, 4get/KN2 mice, B6 Cd36 -/- mice and B6.129P2- Pnpla2tm 1Rze /J ( Pnpla2 -/- ) mice were bred and maintained in specific pathogen free conditions under protocols approved by the institutional animal care committee at Washington University School of Medicine, and were used at 8-12 weeks of age. Lipa +/+ and Lipa -/- mice were maintained at Indiana State University under SPF conditions. In some experiments, mice were injected i.p. with tetrahydrolipistatin (Orlistat; 200 mg/kg, Cayman Chemical) or with 300 µl of 3% thioglycollate (Sigma) immediately prior to IL-4 complexed to mAb anti-IL-4 (IL-4c; containing 5 µg of IL-4, PeproTech, and 25 µg of anti-IL-4 clone 11B11, BioXcell). For infection with H. polygyrus bakeri, we followed protocols described in detail previously. Mice were infected orally by gavage with 200 infe...
Isolation of cells from mice
Peritoneal exudate cells (PECs) were harvested from sacrificed naïve or infected mice by peritoneal lavage with 10 ml of sterile PBS containing 5% FBS (HyClone) per mouse. Total numbers of PECs and peritoneal macrophages were determined by cell counting in combination with multi-color flow cytometry. For analysis of lymphocyte activation by flow cytometry, single cell suspensions were prepared from lymphoid organs by forcing them through 70 µm strainers, and washing with RPMI containing 5% FBS (Corning Inc). Red blood cells were lysed with ACK lysis buffer (0.15 M NH 4 Cl, 1 mM KHCO 3, 0.1 mM EDTA). Bone marrow was flushed from the femur and tibia using RPMI containing 5% FBS and dissociated into a single cell suspension by repeated pipetting. Cells were maintained on ice until use or analysis.
Preparation of macrophages from bone marrow and macrophage activation
Bone marrow cells were differentiated in the presence of recombinant mouse M-CSF (20 ng/ml; R&D Systems) in complete medium (RPMI 1640 containing 10 mM glucose, 2 mM L-glutamine, 100 U/mL of penicillin/streptomycin and 10% FBS) for 7 days. Day 7 macrophages were washed and variously stimulated with IL-4 (20 ng/ml; PeproTech) or lipopolysaccharide (LPS, 20 ng/ml; Sigma) plus IFN-γ (50 ng/ml; R&D Systems) in the presence or absence of 200 µM etomoxir (ETO; Sigma), 20 µM sulfo-n-succinimidyl oleate (SSO), 30 µM chloroquine (CLQ; Sigma), 0.1 µM bafilomycin A1 (Baf; Tocris Bioscience), 100 µM Orlistat (Cayman), 20 µM 5-(Tetradecyloxy)-2-furoic Acid (TOFA, Sigma), 5 or 50 µg/ml of LDL (Kalen Biomedical, LLC) and VLDL (Kalen Biomedical, LLC) for 24 h. Macrophages were then harvested and analyzed by flow cytometry for expression of markers of M1 or M2 a...
Isolation of mononuclear cells from human peripheral blood and human macrophage activation
Monocytes were purified from PBMCs using human CD14 MicroBeads (MACS) and then differentiated into macrophages in complete RPMI medium containing 20 ng/ml recombinant human M-CSF (R&D Systems) for 8 days. Human macrophages were harvested on day 8, and stimulated with 20 ng/ml human IL-4 (R&D Systems) or 50 ng/ml human IFN-γ (R&D Systems) plus 20 ng/ml LPS for 24 h.
Retroviral transduction
Retroviral transduction of macrophages was accomplished using a protocol that we have used previously to transduce bone marrow derived dendritic cells. Sequences for luciferase and for Lipa short hairpin RNAs (shRNAs) were obtained from Open Biosystems and cloned into the MSCV-LTRmir30-PI8 retroviral vector, encoding huCD8 as a reporter. Two independent sequences were used to target Lipa: TCAAGTCCAGCATTCACTG, and TGTGCTTCAGAGACCAGGT. Recombinant retroviruses were used for spin infection (800 x g, 2 h) of day 3 bone marrow macrophage cultures. After 7 days in culture with M-CSF, macrophages were harvested and transduced macrophages were gated based on huCD8 expression. Both shRNAs were used for experiments, with similar results, but only data from experiments that utilized TGTGCTTCAGAGACCAGGT are shown. For the bone marrow chimera, bone marrow cells from donor mice were collected and...
Transmission electron microscopy
For ultrastructural analysis, cells were fixed in 2% paraformaldehyde, 2.5% glutaraldehyde (Polysciences Inc.), 0.05% malachite green (Sigma) in 100 mM sodium cacodylate buffer, pH 7.2 for 1 h at 22°C. The malachite green was incorporated into the fixative to stabilize lipid constituents soluble in aqueous glutaraldehyde. Samples were washed in cacodylate buffer and post-fixed in 1% osmium tetroxide (Polysciences Inc.) for 1 h. Samples were then rinsed extensively in distilled H 2 O prior to en bloc staining with 1% aqueous uranyl acetate (Ted Pella Inc.) for 1 h. Following several rinses in dH 2 O, samples were dehydrated in a graded series of ethanol and embedded in Eponate 12 resin (Ted Pella Inc.). Sections of 95 nm were cut with a Leica Ultracut UC7 ultramicrotome (Leica Microsystems Inc.), stained with uranyl acetate and lead citrate, and viewed on a JEOL 1200 EX transmissi...
Measurement outputs
What raw and processed outputs should exist?
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 endpoint described in the methods before moving into any statistical comparison.
inferred from protocolStatistical comparison
Statistical method not yet structured for this page.
inferred from protocolReporting output
Report representative outputs alongside the summary comparison used to support the conclusion.
inferred from protocolSource and audit
What supports the facts on this page?
Evidence quotes (6)
C57BL/6J mice, 4get/KN2 mice, B6 Cd36 -/- mice and B6.129P2- Pnpla2tm 1Rze /J ( Pnpla2 -/- ) mice were bred and maintained in specific pathogen free conditions under protocols approved by the institutional animal care committee at Washington University School of Medicine, and were used at 8-12 weeks of age. Lipa +/+ and Lipa -/- mice were maintained at Indiana State University under SPF conditions. In some experiments, mice were injected i.p. with tetrahydrolipistatin (Orlistat; 200 mg/kg, Cayman Chemical) or with 300 µl of 3% thioglycollate (Sigma) immediately prior to IL-4 complexed to mAb anti-IL-4 (IL-4c; containing 5 µg of IL-4, PeproTech, and 25 µg of anti-IL-4 clone 11B11, BioXcell). For infection with H. polygyrus bakeri, we followed protocols described in detail previously. Mice were infected orally by gavage with 200 infective L3 stage larvae. For secondary infection experiments, adult H. polygyrus were eliminated from infected mice by oral administration of pyrantel pamoate (1 mg/mouse; Columbia Laboratory) at day 14 post-primary infection, and > 5 weeks later the mice were challenge-infected with 200 L3 stage larv...
Peritoneal exudate cells (PECs) were harvested from sacrificed naïve or infected mice by peritoneal lavage with 10 ml of sterile PBS containing 5% FBS (HyClone) per mouse. Total numbers of PECs and peritoneal macrophages were determined by cell counting in combination with multi-color flow cytometry. For analysis of lymphocyte activation by flow cytometry, single cell suspensions were prepared from lymphoid organs by forcing them through 70 µm strainers, and washing with RPMI containing 5% FBS (Corning Inc). Red blood cells were lysed with ACK lysis buffer (0.15 M NH 4 Cl, 1 mM KHCO 3, 0.1 mM EDTA). Bone marrow was flushed from the femur and tibia using RPMI containing 5% FBS and dissociated into a single cell suspension by repeated pipetting. Cells were maintained on ice until use or analysis.
Bone marrow cells were differentiated in the presence of recombinant mouse M-CSF (20 ng/ml; R&D Systems) in complete medium (RPMI 1640 containing 10 mM glucose, 2 mM L-glutamine, 100 U/mL of penicillin/streptomycin and 10% FBS) for 7 days. Day 7 macrophages were washed and variously stimulated with IL-4 (20 ng/ml; PeproTech) or lipopolysaccharide (LPS, 20 ng/ml; Sigma) plus IFN-γ (50 ng/ml; R&D Systems) in the presence or absence of 200 µM etomoxir (ETO; Sigma), 20 µM sulfo-n-succinimidyl oleate (SSO), 30 µM chloroquine (CLQ; Sigma), 0.1 µM bafilomycin A1 (Baf; Tocris Bioscience), 100 µM Orlistat (Cayman), 20 µM 5-(Tetradecyloxy)-2-furoic Acid (TOFA, Sigma), 5 or 50 µg/ml of LDL (Kalen Biomedical, LLC) and VLDL (Kalen Biomedical, LLC) for 24 h. Macrophages were then harvested and analyzed by flow cytometry for expression of markers of M1 or M2 activation. In some experiments the survival of macrophages was monitored following M2 or M1 activation. For these experiments, bone marrow-derived macrophages (0.5 × 10 6 cells per well of a 48-well plate) were cultured in complete medium with mouse M-CSF (20 ng/ml) and stimulated with IL-4 or...
Monocytes were purified from PBMCs using human CD14 MicroBeads (MACS) and then differentiated into macrophages in complete RPMI medium containing 20 ng/ml recombinant human M-CSF (R&D Systems) for 8 days. Human macrophages were harvested on day 8, and stimulated with 20 ng/ml human IL-4 (R&D Systems) or 50 ng/ml human IFN-γ (R&D Systems) plus 20 ng/ml LPS for 24 h.
Retroviral transduction of macrophages was accomplished using a protocol that we have used previously to transduce bone marrow derived dendritic cells. Sequences for luciferase and for Lipa short hairpin RNAs (shRNAs) were obtained from Open Biosystems and cloned into the MSCV-LTRmir30-PI8 retroviral vector, encoding huCD8 as a reporter. Two independent sequences were used to target Lipa: TCAAGTCCAGCATTCACTG, and TGTGCTTCAGAGACCAGGT. Recombinant retroviruses were used for spin infection (800 x g, 2 h) of day 3 bone marrow macrophage cultures. After 7 days in culture with M-CSF, macrophages were harvested and transduced macrophages were gated based on huCD8 expression. Both shRNAs were used for experiments, with similar results, but only data from experiments that utilized TGTGCTTCAGAGACCAGGT are shown. For the bone marrow chimera, bone marrow cells from donor mice were collected and cultured in complete RPMI medium containing 10 ng/ml IL-3, 10 ng/ml IL-6 and 50 ng/ml murine c-kit ligand for 24 h. Cells were then transduced with retroviral shRNAs targeting Lipa or luciferase, and 5 × 10 5 transduced cells were intravenously injected into irradiated recipients. Recipients w...
For ultrastructural analysis, cells were fixed in 2% paraformaldehyde, 2.5% glutaraldehyde (Polysciences Inc.), 0.05% malachite green (Sigma) in 100 mM sodium cacodylate buffer, pH 7.2 for 1 h at 22°C. The malachite green was incorporated into the fixative to stabilize lipid constituents soluble in aqueous glutaraldehyde. Samples were washed in cacodylate buffer and post-fixed in 1% osmium tetroxide (Polysciences Inc.) for 1 h. Samples were then rinsed extensively in distilled H 2 O prior to en bloc staining with 1% aqueous uranyl acetate (Ted Pella Inc.) for 1 h. Following several rinses in dH 2 O, samples were dehydrated in a graded series of ethanol and embedded in Eponate 12 resin (Ted Pella Inc.). Sections of 95 nm were cut with a Leica Ultracut UC7 ultramicrotome (Leica Microsystems Inc.), stained with uranyl acetate and lead citrate, and viewed on a JEOL 1200 EX transmission electron microscope (JEOL USA Inc.) equipped with an AMT 8 megapixel digital camera (Advanced Microscopy Techniques).
Machine-readable layer
[
{
"@context": "https://schema.org",
"@type": "HowTo",
"name": "Cell-intrinsic lysosomal lipolysis is essential for macrophage alternative activation methods",
"description": "Protocol for Cell-intrinsic lysosomal lipolysis is essential for macrophage alternative activation methods",
"step": [
{
"@type": "HowToStep",
"position": 1,
"name": "Methods",
"text": "C57BL/6J mice, 4get/KN2 mice, B6 Cd36 -/- mice and B6.129P2- Pnpla2tm 1Rze /J ( Pnpla2 -/- ) mice were bred and maintained in specific pathogen free conditions under protocols approved by the institutional animal care committee at Washington University School of Medicine, and were used at 8-12 weeks of age. Lipa +/+ and Lipa -/- mice were maintained at Indiana State University under SPF conditions. In some experiments, mice were injected i.p. with tetrahydrolipistatin (Orlistat; 200 mg/kg, Cayman Chemical) or with 300 µl of 3% thioglycollate (Sigma) immediately prior to IL-4 complexed to mAb anti-IL-4 (IL-4c; containing 5 µg of IL-4, PeproTech, and 25 µg of anti-IL-4 clone 11B11, BioXcell). For infection with H. polygyrus bakeri, we followed protocols described in detail previously. Mice were infected orally by gavage with 200 infe..."
},
{
"@type": "HowToStep",
"position": 2,
"name": "Isolation of cells from mice",
"text": "Peritoneal exudate cells (PECs) were harvested from sacrificed naïve or infected mice by peritoneal lavage with 10 ml of sterile PBS containing 5% FBS (HyClone) per mouse. Total numbers of PECs and peritoneal macrophages were determined by cell counting in combination with multi-color flow cytometry. For analysis of lymphocyte activation by flow cytometry, single cell suspensions were prepared from lymphoid organs by forcing them through 70 µm strainers, and washing with RPMI containing 5% FBS (Corning Inc). Red blood cells were lysed with ACK lysis buffer (0.15 M NH 4 Cl, 1 mM KHCO 3, 0.1 mM EDTA). Bone marrow was flushed from the femur and tibia using RPMI containing 5% FBS and dissociated into a single cell suspension by repeated pipetting. Cells were maintained on ice until use or analysis."
},
{
"@type": "HowToStep",
"position": 3,
"name": "Preparation of macrophages from bone marrow and macrophage activation",
"text": "Bone marrow cells were differentiated in the presence of recombinant mouse M-CSF (20 ng/ml; R&D Systems) in complete medium (RPMI 1640 containing 10 mM glucose, 2 mM L-glutamine, 100 U/mL of penicillin/streptomycin and 10% FBS) for 7 days. Day 7 macrophages were washed and variously stimulated with IL-4 (20 ng/ml; PeproTech) or lipopolysaccharide (LPS, 20 ng/ml; Sigma) plus IFN-γ (50 ng/ml; R&D Systems) in the presence or absence of 200 µM etomoxir (ETO; Sigma), 20 µM sulfo-n-succinimidyl oleate (SSO), 30 µM chloroquine (CLQ; Sigma), 0.1 µM bafilomycin A1 (Baf; Tocris Bioscience), 100 µM Orlistat (Cayman), 20 µM 5-(Tetradecyloxy)-2-furoic Acid (TOFA, Sigma), 5 or 50 µg/ml of LDL (Kalen Biomedical, LLC) and VLDL (Kalen Biomedical, LLC) for 24 h. Macrophages were then harvested and analyzed by flow cytometry for expression of markers of M1 or M2 a..."
},
{
"@type": "HowToStep",
"position": 4,
"name": "Isolation of mononuclear cells from human peripheral blood and human macrophage activation",
"text": "Monocytes were purified from PBMCs using human CD14 MicroBeads (MACS) and then differentiated into macrophages in complete RPMI medium containing 20 ng/ml recombinant human M-CSF (R&D Systems) for 8 days. Human macrophages were harvested on day 8, and stimulated with 20 ng/ml human IL-4 (R&D Systems) or 50 ng/ml human IFN-γ (R&D Systems) plus 20 ng/ml LPS for 24 h."
},
{
"@type": "HowToStep",
"position": 5,
"name": "Retroviral transduction",
"text": "Retroviral transduction of macrophages was accomplished using a protocol that we have used previously to transduce bone marrow derived dendritic cells. Sequences for luciferase and for Lipa short hairpin RNAs (shRNAs) were obtained from Open Biosystems and cloned into the MSCV-LTRmir30-PI8 retroviral vector, encoding huCD8 as a reporter. Two independent sequences were used to target Lipa: TCAAGTCCAGCATTCACTG, and TGTGCTTCAGAGACCAGGT. Recombinant retroviruses were used for spin infection (800 x g, 2 h) of day 3 bone marrow macrophage cultures. After 7 days in culture with M-CSF, macrophages were harvested and transduced macrophages were gated based on huCD8 expression. Both shRNAs were used for experiments, with similar results, but only data from experiments that utilized TGTGCTTCAGAGACCAGGT are shown. For the bone marrow chimera, bone marrow cells from donor mice were collected and..."
},
{
"@type": "HowToStep",
"position": 6,
"name": "Transmission electron microscopy",
"text": "For ultrastructural analysis, cells were fixed in 2% paraformaldehyde, 2.5% glutaraldehyde (Polysciences Inc.), 0.05% malachite green (Sigma) in 100 mM sodium cacodylate buffer, pH 7.2 for 1 h at 22°C. The malachite green was incorporated into the fixative to stabilize lipid constituents soluble in aqueous glutaraldehyde. Samples were washed in cacodylate buffer and post-fixed in 1% osmium tetroxide (Polysciences Inc.) for 1 h. Samples were then rinsed extensively in distilled H 2 O prior to en bloc staining with 1% aqueous uranyl acetate (Ted Pella Inc.) for 1 h. Following several rinses in dH 2 O, samples were dehydrated in a graded series of ethanol and embedded in Eponate 12 resin (Ted Pella Inc.). Sections of 95 nm were cut with a Leica Ultracut UC7 ultramicrotome (Leica Microsystems Inc.), stained with uranyl acetate and lead citrate, and viewed on a JEOL 1200 EX transmissi..."
}
],
"tool": [],
"supply": [],
"isBasedOn": {
"@type": "ScholarlyArticle",
"headline": "Cell-intrinsic lysosomal lipolysis is essential for macrophage alternative activation",
"datePublished": "2014",
"author": [
{
"@type": "Person",
"name": "Stanley Ching-Cheng Huang"
},
{
"@type": "Person",
"name": "Bart Everts"
},
{
"@type": "Person",
"name": "Yulia Ivanova"
},
{
"@type": "Person",
"name": "David O'Sullivan"
},
{
"@type": "Person",
"name": "Marcia Nascimento"
},
{
"@type": "Person",
"name": "Amber M. Smith"
},
{
"@type": "Person",
"name": "Wandy Beatty"
},
{
"@type": "Person",
"name": "Latisha Love-Gregory"
},
{
"@type": "Person",
"name": "Wing Y. Lam"
},
{
"@type": "Person",
"name": "Christina M. O'Neill"
},
{
"@type": "Person",
"name": "Cong Yan"
},
{
"@type": "Person",
"name": "Hong Du"
},
{
"@type": "Person",
"name": "Nada A. Abumrad"
},
{
"@type": "Person",
"name": "Joseph F. Urban"
},
{
"@type": "Person",
"name": "Maxim N. Artyomov"
},
{
"@type": "Person",
"name": "Erika L. Pearce"
},
{
"@type": "Person",
"name": "Edward J. Pearce"
}
],
"identifier": "10.1038/ni.2956"
}
},
{
"@context": "https://schema.org",
"@type": "BreadcrumbList",
"itemListElement": [
{
"@type": "ListItem",
"position": 1,
"name": "Experiments",
"item": "https://replicatescience.com/experiments"
},
{
"@type": "ListItem",
"position": 2,
"name": "Cell-intrinsic lysosomal lipolysis is essential for macrophage alternative activation methods",
"item": "https://replicatescience.com/experiments/cell-intrinsic-lysosomal-lipolysis-is-essential-for-macrophage-alternative-activation-methods-stanley-ching-cheng-huang-pmc4139419/cell-intrinsic-lysosomal-lipolysis-is-essential-for-alternative-activation-of-macrophages-mlph22n6"
}
]
}
]