Impaired immune surveillance accelerates accumulation of senescent cells and aging methods
Aim. Evidence-backed execution summary for Impaired immune surveillance accelerates accumulation of senescent cells and aging methods from Impaired immune surveillance accelerates accumulation of senescent cells and aging.
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?
mouse
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Perforin deficiency drives age-dependent chronic inflammation
reagent used in the protocol.
- Use
- Chronic inflammation is one of the characteristics of aging that are associated with the presence of senescent cells,,. We evaluated the expression of known SASP components in the liver tissue of young, adult, and old WT and Prf1 -/- mice by qPCR analysis (Fig. ). The expression of Il-6, RANTES,...
Perforin deficiency drives age-dependent chronic inflammation
reagent used in the protocol.
- Use
- To determine whether the increased accumulation of senescent cells and immune cells in tissues of Prf1 -/- mice are accompanied by systemic inflammation, we used a cytokine array to compare their serum cytokine levels with those of WT mice at the age of 2, 12, and 24 months. Interestingly, at the age of...
ABT-737 alleviates age-related phenotype of Prf1 -/- mice
reagent used in the protocol.
- Use
- Clearance of p16 Ink4a -positive senescent cells in mice extends their health-span,. To examine whether clearance of senescent cells could counteract age-related phenotype caused by impaired immune cell cytotoxicity, we administered the previously reported senolytic drug ABT-737. Senescent cell viability is depen...
ABT-737 alleviates age-related phenotype of Prf1 -/- mice
reagent used in the protocol.
- Use
- Effects observed in SASP factors point to the possibility of global shift of the transcriptional profile to a younger state following the ABT-737 treatment. Indeed, 74% of genes affected by ABT-737 treatment were also affected by aging (Fig., upper panel). This significantly larger-than-random (Hypergeometric...
ABT-737 extends the lifespan of progeroid mice
reagent used in the protocol.
- Use
- In some cases of accelerated aging, senescent cell formation can be enhanced by a repetitive intrinsic stress; that appears to occur in Hutchinson-Gilford progeria syndrome (HGPS), where accumulation of the progerin protein causes defects of the nuclear lamina and frequent damage to DNA,. LMNA +/G609G mouse...
ABT-737 extends the lifespan of progeroid mice
reagent used in the protocol.
- Use
- The accelerated aging phenotype in LMNA +/G609G mice has been previously linked to an NF-κB-mediated systemic inflammatory response, similarly observed in Prf1 -/- mice. To determine whether the reduced tissue burden of senescent cells following ABT-737 treatment is associated with reduced inflamma...
Perforin deficiency shortens the lifespan of progeroid mice
reagent used in the protocol.
- Use
- The involvement of immune cell cytotoxicity in the regulation of the presence of senescent cells and in accelerated aging in progeroid mice is largely unknown. By crossing Prf1 -/- mice with LMNA +/G609G mice, we generated LMNA +/G609G /Prf1 -/- mice, LMNA +/G609G /Prf1 +/ - mice, and L...
Elimination of senescent cells in vivo
reagent used in the protocol.
- Use
- For selective elimination of senescent cells, 18-month-old Prf1 -/- mice were subjected to ABT-737 treatment for 2 months. At the beginning of each month, 2 consecutive daily intra-peritoneal (i.p.) injection of ABT-737 (25 mg/kg body weight; Selleck Chemicals) or Vehicle as a control were given as...
ABT-737 alleviates age-related phenotype of Prf1 -/- mice
Clearance of p16 Ink4a -positive senescent cells in mice extends their health-span,. To examine whether clearance of senescent cells could counteract age-related phenotype caused by impaired immune cell cytotoxicity, we administered the previously reported senolytic drug ABT-737. Senescent cell viability is depen...
- Use
- Clearance of p16 Ink4a -positive senescent cells in mice extends their health-span,. To examine whether clearance of senescent cells could counteract age-related phenotype caused by impaired immune cell cytotoxicity, we administered the previously reported senolytic drug ABT-737. Senescent cell viability is depen...
Methods
All mice were bred and maintained under specific pathogen-free conditions at the Weizmann Institute of Science in accordance with national animal care guidelines. All animal experiments were approved by the Weizmann Institute's Institutional Animal Care and Use Committee. Mice were maintained under a specific...
- Use
- All mice were bred and maintained under specific pathogen-free conditions at the Weizmann Institute of Science in accordance with national animal care guidelines. All animal experiments were approved by the Weizmann Institute's Institutional Animal Care and Use Committee. Mice were maintained under a specific...
In vivo phenotyping
Animals weights were monitored on monthly basis. Body composition was determined by EchoMRI (Echo Medical Systems). Voluntary exercise was recorded with a recording running wheel placed in individual cages for a week. Grip strength was assessed by a hang-wire test, which measured the time taken by the mouse to drop...
- Use
- Animals weights were monitored on monthly basis. Body composition was determined by EchoMRI (Echo Medical Systems). Voluntary exercise was recorded with a recording running wheel placed in individual cages for a week. Grip strength was assessed by a hang-wire test, which measured the time taken by the mouse to drop...
Histological analysis
Paraffin-embedded tissue sections were stained with hematoxylin and eosin (H&E) for routine examination or with Sirius Red for visualization of fibrotic deposition. For quantification of fibrosis, at least three whole sections from each mouse were scanned by Laser Scanner Cytometry (CompuCyte). These images were qua...
- Use
- Paraffin-embedded tissue sections were stained with hematoxylin and eosin (H&E) for routine examination or with Sirius Red for visualization of fibrotic deposition. For quantification of fibrosis, at least three whole sections from each mouse were scanned by Laser Scanner Cytometry (CompuCyte). These images were qua...
Histological analysis
The following antibodies were used: γ-H2AX (1:200, Cell Signaling, S139), p15 (1:200, Assay Biotech, C0287), p16 (1:400, Abcam, ab54210), p53 (1:50, Santa Cruz, sc-6243), p53BP1 (1:200, Novus, NB100-304), p65 (1:100, Cell Signaling, c-3033 s), and DcR2 (1:200, Enzo, ADI-AAP-371-E), Alexa647 anti-CD45 (1:1...
- Use
- The following antibodies were used: γ-H2AX (1:200, Cell Signaling, S139), p15 (1:200, Assay Biotech, C0287), p16 (1:400, Abcam, ab54210), p53 (1:50, Santa Cruz, sc-6243), p53BP1 (1:200, Novus, NB100-304), p65 (1:100, Cell Signaling, c-3033 s), and DcR2 (1:200, Enzo, ADI-AAP-371-E), Alexa647 anti-CD45 (1:1...
Flow cytometry and ImageStreamX analysis
Tissues were chopped and dissociated to single-cell suspensions by incubation of samples for 50 min at 37 °C. The dissociation solution contained HBSS X1 (#14025050, Gibco) supplemented with 0.1 mg/ml DNase I (#10104159001, Roche), and 2 mg/ml dispase II (#04942078001, Roche) or 0.5̴...
- Use
- Tissues were chopped and dissociated to single-cell suspensions by incubation of samples for 50 min at 37 °C. The dissociation solution contained HBSS X1 (#14025050, Gibco) supplemented with 0.1 mg/ml DNase I (#10104159001, Roche), and 2 mg/ml dispase II (#04942078001, Roche) or 0.5̴...
Flow cytometry and ImageStreamX analysis
For characterization of immune subsets in the tissues by flow cytometry, the cells were maintened in cold FACS buffer (PBS containing 1% FCS and 0.02% Sodium Azid) throughout all procedure. Cells were incubated with FITC anti-CD45 (#103108, Biolegend), PE anti-NK1.1 (#108708, Biolegend), and Alexa647 anti-CD3 (#1002...
- Use
- For characterization of immune subsets in the tissues by flow cytometry, the cells were maintened in cold FACS buffer (PBS containing 1% FCS and 0.02% Sodium Azid) throughout all procedure. Cells were incubated with FITC anti-CD45 (#103108, Biolegend), PE anti-NK1.1 (#108708, Biolegend), and Alexa647 anti-CD3 (#1002...
Flow cytometry and ImageStreamX analysis
For analysis by ImagestreamX cells were first fixed in 4% PFA for 5 min. Fixed cells were washed once with PBS and then with PBS/1 mM MgCl 2 at pH 5.5. They were then resuspended in 5 ml of freshly prepared X-Gal staining solution,and incubated at 37 °C for 12 h while sealed and pr...
- Use
- For analysis by ImagestreamX cells were first fixed in 4% PFA for 5 min. Fixed cells were washed once with PBS and then with PBS/1 mM MgCl 2 at pH 5.5. They were then resuspended in 5 ml of freshly prepared X-Gal staining solution,and incubated at 37 °C for 12 h while sealed and pr...
Statistical analysis
Software used for acquisition, scoring, statistics, or reporting.
- Use
- Results are presented as means ± SDs. Differences between mouse cohorts were analyzed with a two-tailed Student's t -test; values of P < 0.05 were considered significant. Quantitative differences between positive nuclei in indirect immunofluorescence experiments were analyzed by A...
Before you run
What should be confirmed before execution?
First confirmation
Equipment is listed but no product mappings are linked.
Confirm before execution
This page is backed by a publishable Replication Data Ledger package with zero critical source-verification issues.
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?
Perforin deficiency promotes age-related disorders and death
The progressive decline in cellular function that occurs during aging ultimately affects the fitness on the organism level. For example, old mice, like old humans, tend to be less active than young ones. To evaluate the overall fitness of Prf1 -/- mice we tested their voluntary exercise, as well as their muscle strength and coordination. We found that old Prf1 -/- mice showed a reduction of more than 2-fold in voluntary exercise ( P < 0.05; Fig., Supplementary Figure ), and their grip strength and coordination in a "hang-wire" test were significantly decreased, compared to old WT mice ( P = 0.044; Fig. ). Mammals also tend to lose weight during late stages of life. Old Prf1 -/- mice also suffered from progressive weight loss (Fig. ), which was comprised of both muscle loss and fat loss...
ABT-737 alleviates age-related phenotype of Prf1 -/- mice
Clearance of p16 Ink4a -positive senescent cells in mice extends their health-span,. To examine whether clearance of senescent cells could counteract age-related phenotype caused by impaired immune cell cytotoxicity, we administered the previously reported senolytic drug ABT-737. Senescent cell viability is dependent on the expression of anti-apoptotic proteins from the BCL-2 family -. Accordingly, treatment with their specific inhibitors, such as ABT-737 or ABT-263, skews senescent cells toward apoptosis both in vitro and in vivo -. We administrated ABT-737 (25 mg/kg) or a vehicle solution to 18-months-old Prf1 -/- mice, for two consecutive days monthly for a period of two months (Fig. ). Two weeks after the first ABT-737 injection, voluntary activity of ABT-737 treated mice increased compared to control mice (Fig. ). While the activity...
ABT-737 extends the lifespan of progeroid mice
The accelerated aging phenotype in LMNA +/G609G mice has been previously linked to an NF-κB-mediated systemic inflammatory response, similarly observed in Prf1 -/- mice. To determine whether the reduced tissue burden of senescent cells following ABT-737 treatment is associated with reduced inflammatory profile in old LMNA +/G609G mice, we evaluated the activation levels of NF-κB in the liver. The number of cells that are positive for nuclear p65 was more than two-fold decreased in the ABT-737 treated mice comparing to the control mice ( P = 0.019; Fig. ). To examine the systemic effect of ABT-737 treatment in these mice we assayed cytokines in their serum using a cytokine array (Fig., Supplementary Figure ). Interestingly, the amounts of the eight cytokines that had been among the most strongly upregulated in old Prf1 -/W...
Histological analysis
The following antibodies were used: γ-H2AX (1:200, Cell Signaling, S139), p15 (1:200, Assay Biotech, C0287), p16 (1:400, Abcam, ab54210), p53 (1:50, Santa Cruz, sc-6243), p53BP1 (1:200, Novus, NB100-304), p65 (1:100, Cell Signaling, c-3033 s), and DcR2 (1:200, Enzo, ADI-AAP-371-E), Alexa647 anti-CD45 (1:100, #103124, Biolegend), APC anti-Gr-1 (1:100, #108412, Biolegend), APC anti-B220 (1:100, #103212, Biolegend), Alexa647 anti-NK1.1 (1:100, #108720, Biolegend), Alexa647 anti-CD3 (1:100, #100209, Biolegend). For immunostaining, tissue sections (10 µm) were mounted on slides and air-dried for 15 min, fixed in 4% paraformaldehyde (PFA; Sigma) for 10 min, and permeabilized in 0.2% Triton X-100 for 15 min. Slides were blocked in a solution of 5% goat serum, 1% BSA and 0.2% Triton X-100 for 1 h, then incubated with primary antibody overnight at...
Flow cytometry and ImageStreamX analysis
For characterization of immune subsets in the tissues by flow cytometry, the cells were maintened in cold FACS buffer (PBS containing 1% FCS and 0.02% Sodium Azid) throughout all procedure. Cells were incubated with FITC anti-CD45 (#103108, Biolegend), PE anti-NK1.1 (#108708, Biolegend), and Alexa647 anti-CD3 (#100209, Biolegend) mixture for 30 min in 4 °C. The dilution of these antibodies was 1:100 for the studies. After wash, DAPI was shortly introduced in order to exclude dead cells. Cells were analyzed in a SORP-LSRII instrument (BD Biosciences) and FlowJo v10 software. The cell gating strategy is shown in the Supplementary Figure.
Measurement outputs
What raw and processed outputs should exist?
Chronic inflammation is one of the characteristics of aging that are associated with the presence of senescent cells,,. We evaluated the expression of known SASP components 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
To determine whether the increased accumulation of senescent cells and immune cells in tissues of Prf1 -/- mice are accompanied by systemic inflammation, we used a c...
- 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
The establishment of chronic inflammation, also known as "inflammaging", is a major risk factor for both morbidity and mortality in elderly people,. Owing to the 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
Clearance of p16 Ink4a -positive senescent cells in mice extends their health-span,. To examine whether clearance of senescent cells could counteract age-related phenotype cau...
- 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
Chronic inflammation is one of the characteristics of aging that are associated with the presence of senescent cells,,.
from paperScoring or quantification
Quantify the primary readouts for this experiment: Chronic inflammation is one of the characteristics of aging that are associated with the presence of senescent cells,,. We evaluated the expression of known SASP components i...; To determine whether the increased accumulation of senescent cells and immune cells in tissues of Prf1 -/- mice are accompanied by systemic inflammation, we used a c...; The establishment of chronic inflammation, also known as "inflammaging", is a major risk factor for both morbidity and mortality in elderly people,. Owing to the i...; Clearance of p16 Ink4a -positive senescent cells in mice extends their health-span,. To examine whether clearance of senescent cells could counteract age-related phenotype cau....
from paperStatistical comparison
Chronic inflammation is one of the characteristics of aging that are associated with the presence of senescent cells,,. We evaluated the expression of known SASP components i...; The establishment of chronic inflammation, also known as "inflammaging", is a major risk factor for both morbidity and mortality in elderly people,. Owing to the i...; Clearance of p16 Ink4a -positive senescent cells in mice extends their health-span,. To examine whether clearance of senescent cells could counteract age-related phenotype cau...; In some cases of accelerated aging, senescent cell formation can be enhanced by a repetitive intrinsic stress; that appears to occur in Hutchinson-Gilford progeria syndrom...
from paperReporting output
Report representative outputs alongside summary comparisons for Chronic inflammation is one of the characteristics of aging that are associated with the presence of senescent cells,,. We evaluated the expression of known SASP components i..., To determine whether the increased accumulation of senescent cells and immune cells in tissues of Prf1 -/- mice are accompanied by systemic inflammation, we used a c..., The establishment of chronic inflammation, also known as "inflammaging", is a major risk factor for both morbidity and mortality in elderly people,. Owing to the i..., Clearance of p16 Ink4a -positive senescent cells in mice extends their health-span,. To examine whether clearance of senescent cells could counteract age-related phenotype cau....
inferred from protocolStructured statistical methods
Chronic inflammation is one of the characteristics of aging that are associated with the presence of senescent cells,,. We evaluated the expression of known SASP components i...; The establishment of chronic inflammation, also known as "inflammaging", is a major risk factor for both morbidity and mortality in elderly people,. Owing to the i...; Clearance of p16 Ink4a -positive senescent cells in mice extends their health-span,. To examine whether clearance of senescent cells could counteract age-related phenotype cau...; In some cases of accelerated aging, senescent cell formation can be enhanced by a repetitive intrinsic stress; that appears to occur in Hutchinson-Gilford progeria syndrom...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (5)
The progressive decline in cellular function that occurs during aging ultimately affects the fitness on the organism level. For example, old mice, like old humans, tend to be less active than young ones. To evaluate the overall fitness of Prf1 -/- mice we tested their voluntary exercise, as well as their muscle strength and coordination. We found that old Prf1 -/- mice showed a reduction of more than 2-fold in voluntary exercise ( P < 0.05; Fig., Supplementary Figure ), and their grip strength and coordination in a "hang-wire" test were significantly decreased, compared to old WT mice ( P = 0.044; Fig. ). Mammals also tend to lose weight during late stages of life. Old Prf1 -/- mice also suffered from progressive weight loss (Fig. ), which was comprised of both muscle loss and fat loss (Supplementary Figure ). Another aspect of organism-level fitness reduction is kyphosis, an exaggerated rounding of the spine. Old Prf1 -/- mice exhibited a higher incidence of age-related kyphosis than old WT mice ( P < 0.05; Fig. ). An age-related pathology re...
Clearance of p16 Ink4a -positive senescent cells in mice extends their health-span,. To examine whether clearance of senescent cells could counteract age-related phenotype caused by impaired immune cell cytotoxicity, we administered the previously reported senolytic drug ABT-737. Senescent cell viability is dependent on the expression of anti-apoptotic proteins from the BCL-2 family -. Accordingly, treatment with their specific inhibitors, such as ABT-737 or ABT-263, skews senescent cells toward apoptosis both in vitro and in vivo -. We administrated ABT-737 (25 mg/kg) or a vehicle solution to 18-months-old Prf1 -/- mice, for two consecutive days monthly for a period of two months (Fig. ). Two weeks after the first ABT-737 injection, voluntary activity of ABT-737 treated mice increased compared to control mice (Fig. ). While the activity of the control group decreased over the two months as expected from aged mice, the ABT-737-treated group increased their activity 2-fold at the end of the first month and this increase was retained till the end of the experiment ( P = 0.026; Fig. ). At the end of the two-month per...
The accelerated aging phenotype in LMNA +/G609G mice has been previously linked to an NF-κB-mediated systemic inflammatory response, similarly observed in Prf1 -/- mice. To determine whether the reduced tissue burden of senescent cells following ABT-737 treatment is associated with reduced inflammatory profile in old LMNA +/G609G mice, we evaluated the activation levels of NF-κB in the liver. The number of cells that are positive for nuclear p65 was more than two-fold decreased in the ABT-737 treated mice comparing to the control mice ( P = 0.019; Fig. ). To examine the systemic effect of ABT-737 treatment in these mice we assayed cytokines in their serum using a cytokine array (Fig., Supplementary Figure ). Interestingly, the amounts of the eight cytokines that had been among the most strongly upregulated in old Prf1 -/- mice were reduced upon ABT-737 treatment in LMNA +/G609G mice (Fig., Fig. ). We next determined the lifespan of LMNA +/G609G mice treated with ABT-737 or with a vehicle. Strikingly, ABT-737-treatment resulted in a significant increase in median survival (377 vs. 353 days, P =...
The following antibodies were used: γ-H2AX (1:200, Cell Signaling, S139), p15 (1:200, Assay Biotech, C0287), p16 (1:400, Abcam, ab54210), p53 (1:50, Santa Cruz, sc-6243), p53BP1 (1:200, Novus, NB100-304), p65 (1:100, Cell Signaling, c-3033 s), and DcR2 (1:200, Enzo, ADI-AAP-371-E), Alexa647 anti-CD45 (1:100, #103124, Biolegend), APC anti-Gr-1 (1:100, #108412, Biolegend), APC anti-B220 (1:100, #103212, Biolegend), Alexa647 anti-NK1.1 (1:100, #108720, Biolegend), Alexa647 anti-CD3 (1:100, #100209, Biolegend). For immunostaining, tissue sections (10 µm) were mounted on slides and air-dried for 15 min, fixed in 4% paraformaldehyde (PFA; Sigma) for 10 min, and permeabilized in 0.2% Triton X-100 for 15 min. Slides were blocked in a solution of 5% goat serum, 1% BSA and 0.2% Triton X-100 for 1 h, then incubated with primary antibody overnight at 4 °C in the blocking solution. The following day the slides were washed twice with 0.2% Triton X-100 and once with PBS, each time for at least 30 min. In case primary antibodies were not fluorescently labeled, samples were incubated with Dylight-549-conjugated secondary antibody (1:...
For characterization of immune subsets in the tissues by flow cytometry, the cells were maintened in cold FACS buffer (PBS containing 1% FCS and 0.02% Sodium Azid) throughout all procedure. Cells were incubated with FITC anti-CD45 (#103108, Biolegend), PE anti-NK1.1 (#108708, Biolegend), and Alexa647 anti-CD3 (#100209, Biolegend) mixture for 30 min in 4 °C. The dilution of these antibodies was 1:100 for the studies. After wash, DAPI was shortly introduced in order to exclude dead cells. Cells were analyzed in a SORP-LSRII instrument (BD Biosciences) and FlowJo v10 software. The cell gating strategy is shown in the Supplementary Figure.
Machine-readable layer
[
{
"@context": "https://schema.org",
"@type": "HowTo",
"name": "Impaired immune surveillance accelerates accumulation of senescent cells and aging methods",
"description": "Evidence-backed execution summary for Impaired immune surveillance accelerates accumulation of senescent cells and aging methods from Impaired immune surveillance accelerates accumulation of senescent cells and aging.",
"totalTime": "PT466560M",
"step": [
{
"@type": "HowToStep",
"position": 1,
"name": "Perforin deficiency promotes age-related disorders and death",
"text": "The progressive decline in cellular function that occurs during aging ultimately affects the fitness on the organism level. For example, old mice, like old humans, tend to be less active than young ones. To evaluate the overall fitness of Prf1 -/- mice we tested their voluntary exercise, as well as their muscle strength and coordination. We found that old Prf1 -/- mice showed a reduction of more than 2-fold in voluntary exercise ( P < 0.05; Fig., Supplementary Figure ), and their grip strength and coordination in a \"hang-wire\" test were significantly decreased, compared to old WT mice ( P = 0.044; Fig. ). Mammals also tend to lose weight during late stages of life. Old Prf1 -/- mice also suffered from progressive weight loss (Fig. ), which was comprised of both muscle loss and fat loss..."
},
{
"@type": "HowToStep",
"position": 2,
"name": "ABT-737 alleviates age-related phenotype of Prf1 -/- mice",
"text": "Clearance of p16 Ink4a -positive senescent cells in mice extends their health-span,. To examine whether clearance of senescent cells could counteract age-related phenotype caused by impaired immune cell cytotoxicity, we administered the previously reported senolytic drug ABT-737. Senescent cell viability is dependent on the expression of anti-apoptotic proteins from the BCL-2 family -. Accordingly, treatment with their specific inhibitors, such as ABT-737 or ABT-263, skews senescent cells toward apoptosis both in vitro and in vivo -. We administrated ABT-737 (25 mg/kg) or a vehicle solution to 18-months-old Prf1 -/- mice, for two consecutive days monthly for a period of two months (Fig. ). Two weeks after the first ABT-737 injection, voluntary activity of ABT-737 treated mice increased compared to control mice (Fig. ). While the activity..."
},
{
"@type": "HowToStep",
"position": 3,
"name": "ABT-737 extends the lifespan of progeroid mice",
"text": "The accelerated aging phenotype in LMNA +/G609G mice has been previously linked to an NF-κB-mediated systemic inflammatory response, similarly observed in Prf1 -/- mice. To determine whether the reduced tissue burden of senescent cells following ABT-737 treatment is associated with reduced inflammatory profile in old LMNA +/G609G mice, we evaluated the activation levels of NF-κB in the liver. The number of cells that are positive for nuclear p65 was more than two-fold decreased in the ABT-737 treated mice comparing to the control mice ( P = 0.019; Fig. ). To examine the systemic effect of ABT-737 treatment in these mice we assayed cytokines in their serum using a cytokine array (Fig., Supplementary Figure ). Interestingly, the amounts of the eight cytokines that had been among the most strongly upregulated in old Prf1 -/W..."
},
{
"@type": "HowToStep",
"position": 4,
"name": "Histological analysis",
"text": "The following antibodies were used: γ-H2AX (1:200, Cell Signaling, S139), p15 (1:200, Assay Biotech, C0287), p16 (1:400, Abcam, ab54210), p53 (1:50, Santa Cruz, sc-6243), p53BP1 (1:200, Novus, NB100-304), p65 (1:100, Cell Signaling, c-3033 s), and DcR2 (1:200, Enzo, ADI-AAP-371-E), Alexa647 anti-CD45 (1:100, #103124, Biolegend), APC anti-Gr-1 (1:100, #108412, Biolegend), APC anti-B220 (1:100, #103212, Biolegend), Alexa647 anti-NK1.1 (1:100, #108720, Biolegend), Alexa647 anti-CD3 (1:100, #100209, Biolegend). For immunostaining, tissue sections (10 µm) were mounted on slides and air-dried for 15 min, fixed in 4% paraformaldehyde (PFA; Sigma) for 10 min, and permeabilized in 0.2% Triton X-100 for 15 min. Slides were blocked in a solution of 5% goat serum, 1% BSA and 0.2% Triton X-100 for 1 h, then incubated with primary antibody overnight at..."
},
{
"@type": "HowToStep",
"position": 5,
"name": "Flow cytometry and ImageStreamX analysis",
"text": "For characterization of immune subsets in the tissues by flow cytometry, the cells were maintened in cold FACS buffer (PBS containing 1% FCS and 0.02% Sodium Azid) throughout all procedure. Cells were incubated with FITC anti-CD45 (#103108, Biolegend), PE anti-NK1.1 (#108708, Biolegend), and Alexa647 anti-CD3 (#100209, Biolegend) mixture for 30 min in 4 °C. The dilution of these antibodies was 1:100 for the studies. After wash, DAPI was shortly introduced in order to exclude dead cells. Cells were analyzed in a SORP-LSRII instrument (BD Biosciences) and FlowJo v10 software. The cell gating strategy is shown in the Supplementary Figure."
}
],
"tool": [
{
"@type": "HowToTool",
"name": "ABT-737 alleviates age-related phenotype of Prf1 -/- mice"
},
{
"@type": "HowToTool",
"name": "Methods"
},
{
"@type": "HowToTool",
"name": "In vivo phenotyping"
},
{
"@type": "HowToTool",
"name": "Histological analysis"
},
{
"@type": "HowToTool",
"name": "Histological analysis"
},
{
"@type": "HowToTool",
"name": "Flow cytometry and ImageStreamX analysis"
},
{
"@type": "HowToTool",
"name": "Flow cytometry and ImageStreamX analysis"
},
{
"@type": "HowToTool",
"name": "Flow cytometry and ImageStreamX analysis"
}
],
"supply": [
{
"@type": "HowToSupply",
"name": "Perforin deficiency drives age-dependent chronic inflammation"
},
{
"@type": "HowToSupply",
"name": "Perforin deficiency drives age-dependent chronic inflammation"
},
{
"@type": "HowToSupply",
"name": "ABT-737 alleviates age-related phenotype of Prf1 -/- mice"
},
{
"@type": "HowToSupply",
"name": "ABT-737 alleviates age-related phenotype of Prf1 -/- mice"
},
{
"@type": "HowToSupply",
"name": "ABT-737 extends the lifespan of progeroid mice"
},
{
"@type": "HowToSupply",
"name": "ABT-737 extends the lifespan of progeroid mice"
},
{
"@type": "HowToSupply",
"name": "Perforin deficiency shortens the lifespan of progeroid mice"
},
{
"@type": "HowToSupply",
"name": "Elimination of senescent cells in vivo"
}
],
"isBasedOn": {
"@type": "ScholarlyArticle",
"headline": "Impaired immune surveillance accelerates accumulation of senescent cells and aging",
"datePublished": "2018",
"author": [
{
"@type": "Person",
"name": "Yossi Ovadya"
},
{
"@type": "Person",
"name": "Tomer Landsberger"
},
{
"@type": "Person",
"name": "Hanna Leins"
},
{
"@type": "Person",
"name": "Ezra Vadai"
},
{
"@type": "Person",
"name": "Hilah Gal"
},
{
"@type": "Person",
"name": "Anat Biran"
},
{
"@type": "Person",
"name": "Reut Yosef"
},
{
"@type": "Person",
"name": "Adi Sagiv"
},
{
"@type": "Person",
"name": "Amit Agrawal"
},
{
"@type": "Person",
"name": "Alon Shapira"
},
{
"@type": "Person",
"name": "Joseph Windheim"
},
{
"@type": "Person",
"name": "Michael Tsoory"
},
{
"@type": "Person",
"name": "Reinhold Schirmbeck"
},
{
"@type": "Person",
"name": "Ido Amit"
},
{
"@type": "Person",
"name": "Hartmut Geiger"
},
{
"@type": "Person",
"name": "Valery Krizhanovsky"
}
],
"identifier": "10.1038/s41467-018-07825-3"
}
},
{
"@context": "https://schema.org",
"@type": "BreadcrumbList",
"itemListElement": [
{
"@type": "ListItem",
"position": 1,
"name": "Experiments",
"item": "https://replicatescience.com/experiments"
},
{
"@type": "ListItem",
"position": 2,
"name": "Impaired immune surveillance accelerates accumulation of senescent cells and aging methods",
"item": "https://replicatescience.com/experiments/impaired-immune-surveillance-accelerates-accumulation-of-senescent-cells-and-aging-methods-yossi-ovadya-pmc6303397/impaired-immune-surveillance-accelerates-accumulation-of-senescent-cells-and-aging-mlph7zkc"
}
]
}
]