Acute inflammatory profiles differ with sex and age after spinal cord injury methods
Aim. Evidence-backed execution summary for Acute inflammatory profiles differ with sex and age after spinal cord injury methods from Acute inflammatory profiles differ with sex and age after spinal cord injury.
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mouse
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Immunohistochemistry
reagent used in the protocol.
- Use
- 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...
Flow cytometry
reagent used in the protocol.
- Use
- 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 contain...
Antibody labeling and FACS
reagent used in the protocol.
- Use
- 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...
Antibody labeling and FACS
reagent used in the protocol.
- Use
- 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...
Antibody labeling and FACS
reagent used in the protocol.
- Use
- Myeloid cells isolated from injured spinal cords were analyzed for the proportion of MDMs, microglia, and neutrophils to all CD45 + /CD11b + cells. Similarly, MDM/microglia ratios were used to compare total macrophage profiles. Because tubes were emptied during FACS analysis, we also used absolute cell counts to pre...
Gene expression using NanoString
reagent used in the protocol.
- Use
- MDM and microglial RNA was collected and purified using a commercially available single-cell RNA purification kit (Norgen Biotek; Cat #: 51800; Thorold, ON, CA), and obtained RNA was quantified using Nanodrop (Nanodrop Lite; Thermo Fisher Waltham, MA) and tested for degradation using BioAnalyzer (Agilent Technologie...
MDMs present with higher expression levels of activation- and ROS-associated genes
reagent used in the protocol.
- Use
- First, RNA obtained from MDMs and microglia were compared to validate the sorting specificity. Samples from all sex and age groups were combined and a small subset of genes was separately analyzed as assay controls using T tests to determine the sensitivity of discriminating MDMs from microglia, as well as to valida...
Methods
Male and female mice of ages 4- and 14-month-old received T9 contusion SCI and the proportion of microglia, monocyte-derived macrophages (MDM), and neutrophils surrounding the lesion were determined at 3- and 7-day post-injury (DPI) using flow cytometry. Cell counts of microglia and MDMs were obtained using immunohi...
- Use
- Male and female mice of ages 4- and 14-month-old received T9 contusion SCI and the proportion of microglia, monocyte-derived macrophages (MDM), and neutrophils surrounding the lesion were determined at 3- and 7-day post-injury (DPI) using flow cytometry. Cell counts of microglia and MDMs were obtained using immunohi...
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 contain...
- Use
- 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 contain...
Flow cytometry
In total, n =35 mice were used for flow cytometry experiments split between 3- ( n =23) and 7-DPI ( n =12). For the 3-DPI experiments, female ( n =3, 4-MO and n =5, 14-MO) and male ( n =3, 4-MO and n =6, 14-MO) mice survived until perfusions and were used for flow cytometry experiments. For the 7-DPI experiments, fe...
- Use
- In total, n =35 mice were used for flow cytometry experiments split between 3- ( n =23) and 7-DPI ( n =12). For the 3-DPI experiments, female ( n =3, 4-MO and n =5, 14-MO) and male ( n =3, 4-MO and n =6, 14-MO) mice survived until perfusions and were used for flow cytometry experiments. For the 7-DPI experiments, fe...
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...
- Use
- 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...
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...
- Use
- 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...
Antibody labeling and FACS
Myeloid cells isolated from injured spinal cords were analyzed for the proportion of MDMs, microglia, and neutrophils to all CD45 + /CD11b + cells. Similarly, MDM/microglia ratios were used to compare total macrophage profiles. Because tubes were emptied during FACS analysis, we also used absolute cell counts to pre...
- Use
- Myeloid cells isolated from injured spinal cords were analyzed for the proportion of MDMs, microglia, and neutrophils to all CD45 + /CD11b + cells. Similarly, MDM/microglia ratios were used to compare total macrophage profiles. Because tubes were emptied during FACS analysis, we also used absolute cell counts to pre...
Gene expression using NanoString
MDM and microglial RNA was collected and purified using a commercially available single-cell RNA purification kit (Norgen Biotek; Cat #: 51800; Thorold, ON, CA), and obtained RNA was quantified using Nanodrop (Nanodrop Lite; Thermo Fisher Waltham, MA) and tested for degradation using BioAnalyzer (Agilent Technologie...
- Use
- MDM and microglial RNA was collected and purified using a commercially available single-cell RNA purification kit (Norgen Biotek; Cat #: 51800; Thorold, ON, CA), and obtained RNA was quantified using Nanodrop (Nanodrop Lite; Thermo Fisher Waltham, MA) and tested for degradation using BioAnalyzer (Agilent Technologie...
Gene expression using NanoString
All mice used for flow cytometry experiments at 3-DPI were utilized for NanoString analysis except n =1, 14-MO male mouse due to degraded RNA, as well as n =1 microglia sample from a 14-MO female mouse being below the 25-ng limit. Mice within each variable were collapsed across the opposing variable for analyses. Fo...
- Use
- All mice used for flow cytometry experiments at 3-DPI were utilized for NanoString analysis except n =1, 14-MO male mouse due to degraded RNA, as well as n =1 microglia sample from a 14-MO female mouse being below the 25-ng limit. Mice within each variable were collapsed across the opposing variable for analyses. Fo...
Statistics
Software used for acquisition, scoring, statistics, or reporting.
- Use
- Two-way analysis of variance (ANOVA) was used to test the main effects of sex and age as well as for interactions. Sidak's pairwise comparisons were used as post hoc analysis to test effects between groups when the main effect was found significant at p ≤ 0.05. All stats for flow cytometry and immunohist...
NanoString gene expression
Software used for acquisition, scoring, statistics, or reporting.
- Use
- A general linear model was constructed for a two-way multiple analysis of variance (MANOVA) to test the main effects of sex and age as well as for interactions. Overall effects and main effects of each gene were determined. Estimated marginal means (EMM) and corresponding standard error of measurements were derived...
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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?
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 i...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Male and female mice of ages 4- and 14-month-old received T9 contusion SCI and the proportion of microglia, monocyte-derived macrophages (MDM), and neutrophils surrounding the l...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
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 Biotechnolo...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Myeloid cells isolated from injured spinal cords were analyzed for the proportion of MDMs, microglia, and neutrophils to all CD45 + /CD11b + cells. Similarly, MDM/microglia rati...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
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
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).
from paperScoring or quantification
Quantify the primary readouts for this experiment: 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 i...; Male and female mice of ages 4- and 14-month-old received T9 contusion SCI and the proportion of microglia, monocyte-derived macrophages (MDM), and neutrophils surrounding the l...; 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 Biotechnolo...; Myeloid cells isolated from injured spinal cords were analyzed for the proportion of MDMs, microglia, and neutrophils to all CD45 + /CD11b + cells. Similarly, MDM/microglia rati....
from paperStatistical comparison
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 Biotechnolo...; Two-way analysis of variance (ANOVA) was used to test the main effects of sex and age as well as for interactions. Sidak's pairwise comparisons were used as post hoc analy...; A general linear model was constructed for a two-way multiple analysis of variance (MANOVA) to test the main effects of sex and age as well as for interactions. Overall effects...; While there was no main effect of either sex ( F (1, 10) =77.75, p <0.08; Fig. b) or age ( F (1, 10) =0.25, p <0.925; Fig. c) for microglia, there were 5 of 24 analyzed genes si...
from paperReporting output
Report representative outputs alongside summary comparisons for 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 i..., Male and female mice of ages 4- and 14-month-old received T9 contusion SCI and the proportion of microglia, monocyte-derived macrophages (MDM), and neutrophils surrounding the l..., 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 Biotechnolo..., Myeloid cells isolated from injured spinal cords were analyzed for the proportion of MDMs, microglia, and neutrophils to all CD45 + /CD11b + cells. Similarly, MDM/microglia rati....
inferred from protocolStructured statistical methods
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 Biotechnolo...; Two-way analysis of variance (ANOVA) was used to test the main effects of sex and age as well as for interactions. Sidak's pairwise comparisons were used as post hoc analy...; A general linear model was constructed for a two-way multiple analysis of variance (MANOVA) to test the main effects of sex and age as well as for interactions. Overall effects...; While there was no main effect of either sex ( F (1, 10) =77.75, p <0.08; Fig. b) or age ( F (1, 10) =0.25, p <0.925; Fig. c) for microglia, there were 5 of 24 analyzed genes si...
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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
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"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..."
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"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..."
},
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"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."
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"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..."
}
],
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{
"@type": "HowToTool",
"name": "Methods"
},
{
"@type": "HowToTool",
"name": "Flow cytometry"
},
{
"@type": "HowToTool",
"name": "Flow cytometry"
},
{
"@type": "HowToTool",
"name": "Antibody labeling and FACS"
},
{
"@type": "HowToTool",
"name": "Antibody labeling and FACS"
},
{
"@type": "HowToTool",
"name": "Antibody labeling and FACS"
},
{
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"name": "Gene expression using NanoString"
},
{
"@type": "HowToTool",
"name": "Gene expression using NanoString"
}
],
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{
"@type": "HowToSupply",
"name": "Immunohistochemistry"
},
{
"@type": "HowToSupply",
"name": "Flow cytometry"
},
{
"@type": "HowToSupply",
"name": "Antibody labeling and FACS"
},
{
"@type": "HowToSupply",
"name": "Antibody labeling and FACS"
},
{
"@type": "HowToSupply",
"name": "Antibody labeling and FACS"
},
{
"@type": "HowToSupply",
"name": "Gene expression using NanoString"
},
{
"@type": "HowToSupply",
"name": "MDMs present with higher expression levels of activation- and ROS-associated genes"
}
],
"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"
}
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"name": "Acute inflammatory profiles differ with sex and age after spinal cord injury methods",
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