Acute inflammatory profiles differ with sex and age after spinal cord injury 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?
Immunohistochemistry
Mice were anesthetized using an overdose of ketamine and xylazine as described above. Mice were perfused using cardiac puncture first with 0.1 M PBS, then with 4% formaldehyde in PBS made from paraformaldehyde. The spinal cords were extracted from mice, then post-fixed for 2 h at room temperature and washed in 0.2 M phosphate buffer (PB) overnight. The cords were transferred to a 30% sucrose solution for 1 week before blocking in optimal cutting temperature compound (OCT; Thomas Scientific; Swedesboro, NJ). The spinal cords were blocked in a random order but ensuring a representation of all groups in each block ( n =5/block). Blocks were cut between -18 o C and -20 o C at 10.0-µm thick in the coronal plane. Each block was cut in a series of 10 equal representative sets with 100.0 µm of z -distance between repeated sections. First, one set was stained as previously desc...
Methods
All procedures are approved by the University of Kentucky's Institutional Animal Care and Use Committee. In total, 74 female and male C57Bl/6 mice of ages 4- ( n =33; Jackson Laboratories), and 14-month old (MO; n =41; derived from Jackson Laboratory but aged by the National Institute on Aging) received laminectomy only as a sham injury, or laminectomy with 60- or 75-kDyn SCI under ketamine (100.0 mg/kg) and xylazine (10.0 mg/kg) anesthesia as previously described using the Infinite Horizons Impactor (Precision Systems Instrumentation, LLC, Fairfax Station, VA) [ ]. After SCI, all mice received buprenorphine (Buprenex SR, 1.0 mg/kg day of surgery), as well as an antibiotic (Enrofloxacin, 5.0 mg/kg) dissolved in saline (1.0 mL/day) for up to 5 days post-SCI. Manual bladder expressions were performed 2x/day for the duration of the study. Mice of ages 4- and 14-MO were chosen as a...
Flow cytometry
Mice receiving 75 kDyn SCI were anesthetized at 3- and 7-DPI using an overdose of ketamine and xylazine and were perfused using sterile 0.01 M phosphate-buffered saline (PBS) containing 1.0 mM EDTA. An 8.0-mm section of the spinal cord surrounding the SCI lesion was dissected and placed in a digestion buffer containing a 1:1 solution of RPMI and Accumax (Thermo Fisher Scientific, Waltham, MA). The cords were mashed through a 70-µm diameter meshing into a 30% Percoll solution diluted in RPMI (Thermo Fisher Scientific, Waltham, MA). Cells were centrifuged at 1500 G for 15 min at 10 o C to pellet cells, and the floating myelin layer was aspirated. Myeloid cells and debris were then suspended in red blood cell lysis buffer and pelleted. Cells were washed in PBS and suspended in cell staining buffer (Cat #: 420201; Biolegend, San Diego, CA) for antibody labeling.
Antibody labeling and FACS
Endogenous Fc receptors were blocked for 15 min on ice using neutralizing antibodies targeting CD16/32 (1:100; Fc Block; Cat #: 553142; BD Biosciences, San Jose, CA). Next, cells obtained from the spinal cord were washed and resuspended in an antibody cocktail labeling all cells of the myeloid and lymphocyte lineage (Rat anti-CD45-APC conjugate; 1:100; Cat #: 559864; BD Biosciences, San Jose, CA), as well as for the macrophage-specific integrin marker (Rat anti-CD11b-PE conjugate; 1:200; Cat #: 553311; BD Biosciences, San Jose, CA) and a neutrophil selection marker (Rat anti-Ly6G-PE-Cy7 conjugate; 1:50; Cat #: 560601; BD Biosciences, San Jose, CA). Antibodies were incubated for 1 h on ice before washing and re-suspending in cell staining buffer.
Antibody labeling and FACS
MDMs and microglia were sorted and collected in separate tubes containing cell lysis buffer (Cat #: 51800; Norgen Biotek; Ontario, CAN) using FACS (iCyt sy3200; Sony Biotechnologies; San Jose, CA). FACS was performed by the University of Kentucky's Flow Cytometry and Immune Monitoring Facility. An appropriate gating strategy was applied based on forward and side scatter plots to isolate cells from debris, exclude doublets, and set compensations using single-antibody controls (Fig. a). Experimental controls consisted of no-stain, single-channel, and fluorescence minus one. Cells obtained from 4-MO female mice that were designated for control purposes were used to set compensations. MDMs (CD45 + /CD11b +high /Ly6G - ) and microglia (CD45 + /CD11b +low /Ly6G - ) were identified and sorted into separate tubes. Neutrophils (CD45 + /CD11b + /Ly6G + ) were identified in flow plots and...
Immunohistochemistry
Slides were visualized and analyzed using Halo software (Indica Labs; Albuquerque, NM). In total, 3 sections were used to analyze cell counts of MDMs and microglia at the lesion epicenter as well as at 100-µm rostral and caudal to the epicenter. The region of analysis was limited to within the lesion, and cells were counted using unbiased procedures adapted from fluorescent labeling techniques [ ]. Annotations encompassing the lesion were partitioned into evenly spaced, but randomly placed, 100-µm 2 boxes. MDMs were identified by the presence of brown labeling (F4/80) and all cells containing a nucleus that were within the box, or touching the top or right margins, were counted. Microglia were identified by the presence of black P2y12 labeling which often masked nuclear cresyl violet staining; therefore, a standard criterion was set for counting microglia. All P2y12 labeling...
Immunohistochemistry reveals a male-dependent increase in microglia at 3-DPI
Flow cytometry analyses included the spinal cord tissue encompassing multiple spinal levels (8 mm) [ ] including the entirety of lesion tissue. To better understand the inflammatory response at the level of the lesion epicenter, we generated tissue sections from 4- and 14-MO, male and female mice, with and without SCI (Fig. a, b). To examine both cell types in situ, we sampled injured tissue at the lesion epicenter (sections containing the least amount of spared tissue) and generated cell counts based upon P2y12 labeling of microglia and F4/80 labeling of MDMs. In the absence of injury, P2y12 + microglia were evident with a ramified phenotype (Fig. a). As reported previously, there was little to no staining for F4/80, the pan marker for activated macrophages, in uninjured tissue [ ]. After SCI, F4/80 +, round macrophages were clearly discernable at the lesion epicenter (Fig. ). Micro...
Measurement outputs
What raw and processed outputs should exist?
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: 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 Microscopy-based tissue or cell-state imaging.
inferred from protocolSource and audit
What supports the facts on this page?
Evidence quotes (7)
Mice were anesthetized using an overdose of ketamine and xylazine as described above. Mice were perfused using cardiac puncture first with 0.1 M PBS, then with 4% formaldehyde in PBS made from paraformaldehyde. The spinal cords were extracted from mice, then post-fixed for 2 h at room temperature and washed in 0.2 M phosphate buffer (PB) overnight. The cords were transferred to a 30% sucrose solution for 1 week before blocking in optimal cutting temperature compound (OCT; Thomas Scientific; Swedesboro, NJ). The spinal cords were blocked in a random order but ensuring a representation of all groups in each block ( n =5/block). Blocks were cut between -18 o C and -20 o C at 10.0-µm thick in the coronal plane. Each block was cut in a series of 10 equal representative sets with 100.0 µm of z -distance between repeated sections. First, one set was stained as previously described [ ] using eriochrome cyanine and neurofilament (Chicken anti-NF-200; 1:1,500; Cat #: NFH; Aves Laboratories; Davis, CA) to identify spared tissue. The section containing the least amount of spared tissue was considered the lesion epicenter and was used to standardize analysis with respect to t...
All procedures are approved by the University of Kentucky's Institutional Animal Care and Use Committee. In total, 74 female and male C57Bl/6 mice of ages 4- ( n =33; Jackson Laboratories), and 14-month old (MO; n =41; derived from Jackson Laboratory but aged by the National Institute on Aging) received laminectomy only as a sham injury, or laminectomy with 60- or 75-kDyn SCI under ketamine (100.0 mg/kg) and xylazine (10.0 mg/kg) anesthesia as previously described using the Infinite Horizons Impactor (Precision Systems Instrumentation, LLC, Fairfax Station, VA) [ ]. After SCI, all mice received buprenorphine (Buprenex SR, 1.0 mg/kg day of surgery), as well as an antibiotic (Enrofloxacin, 5.0 mg/kg) dissolved in saline (1.0 mL/day) for up to 5 days post-SCI. Manual bladder expressions were performed 2x/day for the duration of the study. Mice of ages 4- and 14-MO were chosen as a representative shift in average clinical demographics from 27 to 43 years old at the time of SCI since the 1970s [ ].
Mice receiving 75 kDyn SCI were anesthetized at 3- and 7-DPI using an overdose of ketamine and xylazine and were perfused using sterile 0.01 M phosphate-buffered saline (PBS) containing 1.0 mM EDTA. An 8.0-mm section of the spinal cord surrounding the SCI lesion was dissected and placed in a digestion buffer containing a 1:1 solution of RPMI and Accumax (Thermo Fisher Scientific, Waltham, MA). The cords were mashed through a 70-µm diameter meshing into a 30% Percoll solution diluted in RPMI (Thermo Fisher Scientific, Waltham, MA). Cells were centrifuged at 1500 G for 15 min at 10 o C to pellet cells, and the floating myelin layer was aspirated. Myeloid cells and debris were then suspended in red blood cell lysis buffer and pelleted. Cells were washed in PBS and suspended in cell staining buffer (Cat #: 420201; Biolegend, San Diego, CA) for antibody labeling.
Endogenous Fc receptors were blocked for 15 min on ice using neutralizing antibodies targeting CD16/32 (1:100; Fc Block; Cat #: 553142; BD Biosciences, San Jose, CA). Next, cells obtained from the spinal cord were washed and resuspended in an antibody cocktail labeling all cells of the myeloid and lymphocyte lineage (Rat anti-CD45-APC conjugate; 1:100; Cat #: 559864; BD Biosciences, San Jose, CA), as well as for the macrophage-specific integrin marker (Rat anti-CD11b-PE conjugate; 1:200; Cat #: 553311; BD Biosciences, San Jose, CA) and a neutrophil selection marker (Rat anti-Ly6G-PE-Cy7 conjugate; 1:50; Cat #: 560601; BD Biosciences, San Jose, CA). Antibodies were incubated for 1 h on ice before washing and re-suspending in cell staining buffer.
MDMs and microglia were sorted and collected in separate tubes containing cell lysis buffer (Cat #: 51800; Norgen Biotek; Ontario, CAN) using FACS (iCyt sy3200; Sony Biotechnologies; San Jose, CA). FACS was performed by the University of Kentucky's Flow Cytometry and Immune Monitoring Facility. An appropriate gating strategy was applied based on forward and side scatter plots to isolate cells from debris, exclude doublets, and set compensations using single-antibody controls (Fig. a). Experimental controls consisted of no-stain, single-channel, and fluorescence minus one. Cells obtained from 4-MO female mice that were designated for control purposes were used to set compensations. MDMs (CD45 + /CD11b +high /Ly6G - ) and microglia (CD45 + /CD11b +low /Ly6G - ) were identified and sorted into separate tubes. Neutrophils (CD45 + /CD11b + /Ly6G + ) were identified in flow plots and used for cell profiling. Fig. 1 Flow cytometry reveals different inflammatory profiles between sexes at 3-DPI. a Myeloid cells were isolated from the injured spinal cords of male and female, 4- and 14-MO mice at 3-DPI using a consistent gating strategy. b Representative flow plots of cells that were...
Slides were visualized and analyzed using Halo software (Indica Labs; Albuquerque, NM). In total, 3 sections were used to analyze cell counts of MDMs and microglia at the lesion epicenter as well as at 100-µm rostral and caudal to the epicenter. The region of analysis was limited to within the lesion, and cells were counted using unbiased procedures adapted from fluorescent labeling techniques [ ]. Annotations encompassing the lesion were partitioned into evenly spaced, but randomly placed, 100-µm 2 boxes. MDMs were identified by the presence of brown labeling (F4/80) and all cells containing a nucleus that were within the box, or touching the top or right margins, were counted. Microglia were identified by the presence of black P2y12 labeling which often masked nuclear cresyl violet staining; therefore, a standard criterion was set for counting microglia. All P2y12 labeling that presented with a soma and at least 5 µm of continual P2y12 staining, either in diameter or length, were counted. Staining length was assessed using a built-in ruler tool in Halo. Due to differences in sizes between 4- and 14-MO mouse spinal cords, as well as occasional rips and folds in t...
Flow cytometry analyses included the spinal cord tissue encompassing multiple spinal levels (8 mm) [ ] including the entirety of lesion tissue. To better understand the inflammatory response at the level of the lesion epicenter, we generated tissue sections from 4- and 14-MO, male and female mice, with and without SCI (Fig. a, b). To examine both cell types in situ, we sampled injured tissue at the lesion epicenter (sections containing the least amount of spared tissue) and generated cell counts based upon P2y12 labeling of microglia and F4/80 labeling of MDMs. In the absence of injury, P2y12 + microglia were evident with a ramified phenotype (Fig. a). As reported previously, there was little to no staining for F4/80, the pan marker for activated macrophages, in uninjured tissue [ ]. After SCI, F4/80 +, round macrophages were clearly discernable at the lesion epicenter (Fig. ). Microglia took on an amoeboid phenotype with P2y12 labeling punctate and concentrated in and around the nucleus as reported previously [ ]. Using criteria, we established previously for SCI [ ], we classified macrophages as MDMs (F4/80 + only) or microglia (F4/80 + /P2y12 + or P2y12 + only). Fig. 3 Young...
Machine-readable layer
[
{
"@context": "https://schema.org",
"@type": "HowTo",
"name": "Acute inflammatory profiles differ with sex and age after spinal cord injury methods",
"description": "Protocol for Acute inflammatory profiles differ with sex and age after spinal cord injury methods",
"step": [
{
"@type": "HowToStep",
"position": 1,
"name": "Immunohistochemistry",
"text": "Mice were anesthetized using an overdose of ketamine and xylazine as described above. Mice were perfused using cardiac puncture first with 0.1 M PBS, then with 4% formaldehyde in PBS made from paraformaldehyde. The spinal cords were extracted from mice, then post-fixed for 2 h at room temperature and washed in 0.2 M phosphate buffer (PB) overnight. The cords were transferred to a 30% sucrose solution for 1 week before blocking in optimal cutting temperature compound (OCT; Thomas Scientific; Swedesboro, NJ). The spinal cords were blocked in a random order but ensuring a representation of all groups in each block ( n =5/block). Blocks were cut between -18 o C and -20 o C at 10.0-µm thick in the coronal plane. Each block was cut in a series of 10 equal representative sets with 100.0 µm of z -distance between repeated sections. First, one set was stained as previously desc..."
},
{
"@type": "HowToStep",
"position": 2,
"name": "Methods",
"text": "All procedures are approved by the University of Kentucky's Institutional Animal Care and Use Committee. In total, 74 female and male C57Bl/6 mice of ages 4- ( n =33; Jackson Laboratories), and 14-month old (MO; n =41; derived from Jackson Laboratory but aged by the National Institute on Aging) received laminectomy only as a sham injury, or laminectomy with 60- or 75-kDyn SCI under ketamine (100.0 mg/kg) and xylazine (10.0 mg/kg) anesthesia as previously described using the Infinite Horizons Impactor (Precision Systems Instrumentation, LLC, Fairfax Station, VA) [ ]. After SCI, all mice received buprenorphine (Buprenex SR, 1.0 mg/kg day of surgery), as well as an antibiotic (Enrofloxacin, 5.0 mg/kg) dissolved in saline (1.0 mL/day) for up to 5 days post-SCI. Manual bladder expressions were performed 2x/day for the duration of the study. Mice of ages 4- and 14-MO were chosen as a..."
},
{
"@type": "HowToStep",
"position": 3,
"name": "Flow cytometry",
"text": "Mice receiving 75 kDyn SCI were anesthetized at 3- and 7-DPI using an overdose of ketamine and xylazine and were perfused using sterile 0.01 M phosphate-buffered saline (PBS) containing 1.0 mM EDTA. An 8.0-mm section of the spinal cord surrounding the SCI lesion was dissected and placed in a digestion buffer containing a 1:1 solution of RPMI and Accumax (Thermo Fisher Scientific, Waltham, MA). The cords were mashed through a 70-µm diameter meshing into a 30% Percoll solution diluted in RPMI (Thermo Fisher Scientific, Waltham, MA). Cells were centrifuged at 1500 G for 15 min at 10 o C to pellet cells, and the floating myelin layer was aspirated. Myeloid cells and debris were then suspended in red blood cell lysis buffer and pelleted. Cells were washed in PBS and suspended in cell staining buffer (Cat #: 420201; Biolegend, San Diego, CA) for antibody labeling."
},
{
"@type": "HowToStep",
"position": 4,
"name": "Antibody labeling and FACS",
"text": "Endogenous Fc receptors were blocked for 15 min on ice using neutralizing antibodies targeting CD16/32 (1:100; Fc Block; Cat #: 553142; BD Biosciences, San Jose, CA). Next, cells obtained from the spinal cord were washed and resuspended in an antibody cocktail labeling all cells of the myeloid and lymphocyte lineage (Rat anti-CD45-APC conjugate; 1:100; Cat #: 559864; BD Biosciences, San Jose, CA), as well as for the macrophage-specific integrin marker (Rat anti-CD11b-PE conjugate; 1:200; Cat #: 553311; BD Biosciences, San Jose, CA) and a neutrophil selection marker (Rat anti-Ly6G-PE-Cy7 conjugate; 1:50; Cat #: 560601; BD Biosciences, San Jose, CA). Antibodies were incubated for 1 h on ice before washing and re-suspending in cell staining buffer."
},
{
"@type": "HowToStep",
"position": 5,
"name": "Antibody labeling and FACS",
"text": "MDMs and microglia were sorted and collected in separate tubes containing cell lysis buffer (Cat #: 51800; Norgen Biotek; Ontario, CAN) using FACS (iCyt sy3200; Sony Biotechnologies; San Jose, CA). FACS was performed by the University of Kentucky's Flow Cytometry and Immune Monitoring Facility. An appropriate gating strategy was applied based on forward and side scatter plots to isolate cells from debris, exclude doublets, and set compensations using single-antibody controls (Fig. a). Experimental controls consisted of no-stain, single-channel, and fluorescence minus one. Cells obtained from 4-MO female mice that were designated for control purposes were used to set compensations. MDMs (CD45 + /CD11b +high /Ly6G - ) and microglia (CD45 + /CD11b +low /Ly6G - ) were identified and sorted into separate tubes. Neutrophils (CD45 + /CD11b + /Ly6G + ) were identified in flow plots and..."
},
{
"@type": "HowToStep",
"position": 6,
"name": "Immunohistochemistry",
"text": "Slides were visualized and analyzed using Halo software (Indica Labs; Albuquerque, NM). In total, 3 sections were used to analyze cell counts of MDMs and microglia at the lesion epicenter as well as at 100-µm rostral and caudal to the epicenter. The region of analysis was limited to within the lesion, and cells were counted using unbiased procedures adapted from fluorescent labeling techniques [ ]. Annotations encompassing the lesion were partitioned into evenly spaced, but randomly placed, 100-µm 2 boxes. MDMs were identified by the presence of brown labeling (F4/80) and all cells containing a nucleus that were within the box, or touching the top or right margins, were counted. Microglia were identified by the presence of black P2y12 labeling which often masked nuclear cresyl violet staining; therefore, a standard criterion was set for counting microglia. All P2y12 labeling..."
},
{
"@type": "HowToStep",
"position": 7,
"name": "Immunohistochemistry reveals a male-dependent increase in microglia at 3-DPI",
"text": "Flow cytometry analyses included the spinal cord tissue encompassing multiple spinal levels (8 mm) [ ] including the entirety of lesion tissue. To better understand the inflammatory response at the level of the lesion epicenter, we generated tissue sections from 4- and 14-MO, male and female mice, with and without SCI (Fig. a, b). To examine both cell types in situ, we sampled injured tissue at the lesion epicenter (sections containing the least amount of spared tissue) and generated cell counts based upon P2y12 labeling of microglia and F4/80 labeling of MDMs. In the absence of injury, P2y12 + microglia were evident with a ramified phenotype (Fig. a). As reported previously, there was little to no staining for F4/80, the pan marker for activated macrophages, in uninjured tissue [ ]. After SCI, F4/80 +, round macrophages were clearly discernable at the lesion epicenter (Fig. ). Micro..."
}
],
"tool": [],
"supply": [],
"isBasedOn": {
"@type": "ScholarlyArticle",
"headline": "Acute inflammatory profiles differ with sex and age after spinal cord injury",
"datePublished": "2021",
"author": [
{
"@type": "Person",
"name": "Andrew N. Stewart"
},
{
"@type": "Person",
"name": "John L. Lowe"
},
{
"@type": "Person",
"name": "Ethan P. Glaser"
},
{
"@type": "Person",
"name": "Caitlin A. Mott"
},
{
"@type": "Person",
"name": "Ryan K. Shahidehpour"
},
{
"@type": "Person",
"name": "Katelyn E. McFarlane"
},
{
"@type": "Person",
"name": "William M. Bailey"
},
{
"@type": "Person",
"name": "Bei Zhang"
},
{
"@type": "Person",
"name": "John C. Gensel"
}
],
"identifier": "10.1186/s12974-021-02161-8"
}
},
{
"@context": "https://schema.org",
"@type": "BreadcrumbList",
"itemListElement": [
{
"@type": "ListItem",
"position": 1,
"name": "Experiments",
"item": "https://replicatescience.com/experiments"
},
{
"@type": "ListItem",
"position": 2,
"name": "Acute inflammatory profiles differ with sex and age after spinal cord injury methods",
"item": "https://replicatescience.com/experiments/acute-inflammatory-profiles-differ-with-sex-and-age-after-spinal-cord-injury-methods-andrew-n-stewart-pmc8120918/acute-inflammatory-profiles-differ-with-sex-and-age-after-spinal-cord-injury-mlph94lg"
}
]
}
]