Journal: Developmental and comparative immunology

Article Title: Multi-color Flow Cytometry for Evaluating Age-Related Changes in Memory Lymphocyte Subsets in Dogs

doi: 10.1016/j.dci.2018.05.022

Figure Lengend Snippet: Cell surface and intracellular antibodies and markers used in flow cytometry studies

Article Snippet: For intracellular cytokine staining experiments, both mitogen-stimulated and control cells were concurrently incubated with Brefeldin A (B7651, Sigma-Aldrich, Saint Louis, MO, USA) at a dilution of 1 μl/ml in complete media during the 6-hour stimulation period. table ft1 table-wrap mode="anchored" t5 Table 1: caption a7 Antigen/ marker Conjugated fluorochrome Antigen species Antibody clone # Manufacturer Catalogue # Dilution Reference Cell Surface Viability dye Near infra-red N/A N/A Thermo Fisher {"type":"entrez-nucleotide","attrs":{"text":"L34975","term_id":"522218","term_text":"L34975"}} L34975 1:1000 N/A CD4 PE-Cy7 Canine YKIX302.9 eBioscience 25-5040-41 1:10 N/A CD4 PB Canine YKIX302.9 Biorad MCA1038PB 1:10 N/A CD8 PerCp-Cy5.5 Canine YCATE55.9 eBioscience 46-5080-41 1:25 N/A CD28 APC Canine 5B8 eBioscience 17-0282-41 1:10 N/A CD44 FITC Canine YKIX337.8 Thermo Fisher 11-5440-41 1:10 N/A CD62L PE Human FMC46 BioRad MCA1076PET 1:10 ( Bismarck et al., 2012 ; Hartley and Tarleton, 2015 ; Rothe et al., 2017 ; Schuberth et al., 2007 ) CD45RA Biotin Canine CA4.1D3 PFM Lab N/A 1:50 ( Cobbold and Metcalfe, 1994 ; Reis et al., 2005 ) Streptavadin BV711 N/A N/A Biolegend 405241 1:50 N/A CD25 PE Canine P4A10 eBioscience 12-0250-42 1:10 N/A Intracellular CD3 PB Human CD3-12 BioRad MCA1477PB 1:50 ( Monjazeb et al., 2016 ) CD3 FITC Human CD3-12 BioRad MCA1477F 1:50 ( Monjazeb et al., 2016 ) IFNγ PE Bovine CC302 BioRad MCA1783PE 1:10 ( Hartley and Tarleton, 2015 ; Pedersen et al., 2002 ) TNFα BV-785 Human MAB-11 Biolegend 502947 1:25 ( Moreira et al., 2015 ) Ki67 PE-Cy7 Human 20Raj1 eBioscience 25-5699-42 1:50 ( Galkowska et al., 1996 ) Open in a separate window APC, allophycocyanin; BV-711, brilliant violet™711; BV-785, brilliant violet™785; FITC, fluorescein; PB, pacific blue; PE, phycoerythrin; PE-Cy7, PE-cyanine 7; PE-TR, PE-texas red; PerCPCy5.5, peridinin chlorphyll protein-cyanine 5.5; PFM lab, Peter F. Moore laboratory; N/A, not applicable.

Techniques: Flow Cytometry, Marker

Journal: Developmental and comparative immunology

Article Title: Multi-color Flow Cytometry for Evaluating Age-Related Changes in Memory Lymphocyte Subsets in Dogs

doi: 10.1016/j.dci.2018.05.022

Figure Lengend Snippet: Cell surface and intracellular antibodies and markers used in flow cytometry studies

Article Snippet: CD4 , PE-Cy7 , Canine , YKIX302.9 , eBioscience , 25-5040-41 , 1:10 , N/A.

Techniques: Flow Cytometry, Marker

Journal: Developmental and comparative immunology

Article Title: Multi-color Flow Cytometry for Evaluating Age-Related Changes in Memory Lymphocyte Subsets in Dogs

doi: 10.1016/j.dci.2018.05.022

Figure Lengend Snippet: Cell surface and intracellular antibodies and markers used in flow cytometry studies

Article Snippet: For intracellular cytokine staining experiments, both mitogen-stimulated and control cells were concurrently incubated with Brefeldin A (B7651, Sigma-Aldrich, Saint Louis, MO, USA) at a dilution of 1 μl/ml in complete media during the 6-hour stimulation period. table ft1 table-wrap mode="anchored" t5 Table 1: caption a7 Antigen/ marker Conjugated fluorochrome Antigen species Antibody clone # Manufacturer Catalogue # Dilution Reference Cell Surface Viability dye Near infra-red N/A N/A Thermo Fisher {"type":"entrez-nucleotide","attrs":{"text":"L34975","term_id":"522218","term_text":"L34975"}} L34975 1:1000 N/A CD4 PE-Cy7 Canine YKIX302.9 eBioscience 25-5040-41 1:10 N/A CD4 PB Canine YKIX302.9 Biorad MCA1038PB 1:10 N/A CD8 PerCp-Cy5.5 Canine YCATE55.9 eBioscience 46-5080-41 1:25 N/A CD28 APC Canine 5B8 eBioscience 17-0282-41 1:10 N/A CD44 FITC Canine YKIX337.8 Thermo Fisher 11-5440-41 1:10 N/A CD62L PE Human FMC46 BioRad MCA1076PET 1:10 ( Bismarck et al., 2012 ; Hartley and Tarleton, 2015 ; Rothe et al., 2017 ; Schuberth et al., 2007 ) CD45RA Biotin Canine CA4.1D3 PFM Lab N/A 1:50 ( Cobbold and Metcalfe, 1994 ; Reis et al., 2005 ) Streptavadin BV711 N/A N/A Biolegend 405241 1:50 N/A CD25 PE Canine P4A10 eBioscience 12-0250-42 1:10 N/A Intracellular CD3 PB Human CD3-12 BioRad MCA1477PB 1:50 ( Monjazeb et al., 2016 ) CD3 FITC Human CD3-12 BioRad MCA1477F 1:50 ( Monjazeb et al., 2016 ) IFNγ PE Bovine CC302 BioRad MCA1783PE 1:10 ( Hartley and Tarleton, 2015 ; Pedersen et al., 2002 ) TNFα BV-785 Human MAB-11 Biolegend 502947 1:25 ( Moreira et al., 2015 ) Ki67 PE-Cy7 Human 20Raj1 eBioscience 25-5699-42 1:50 ( Galkowska et al., 1996 ) Open in a separate window APC, allophycocyanin; BV-711, brilliant violet™711; BV-785, brilliant violet™785; FITC, fluorescein; PB, pacific blue; PE, phycoerythrin; PE-Cy7, PE-cyanine 7; PE-TR, PE-texas red; PerCPCy5.5, peridinin chlorphyll protein-cyanine 5.5; PFM lab, Peter F. Moore laboratory; N/A, not applicable.

Techniques: Flow Cytometry, Marker

Journal: Developmental and comparative immunology

Article Title: Multi-color Flow Cytometry for Evaluating Age-Related Changes in Memory Lymphocyte Subsets in Dogs

doi: 10.1016/j.dci.2018.05.022

Figure Lengend Snippet: Functionality of memory T cell subsets after ConA stimulation of PBMCs from young and aged dogs. A) Representative scatter plots illustrating TNFα production by CD4+ and CD8+ T cells with EM- and naïve-like phenotypes after 6 hours of ConA stimulation. The example shown is from an aged, overweight male beagle. B) TNFα+ frequencies and TNFα MFI of CD4+ and CD8+ memory subsets. C) TNFα+ frequencies of CD4+ and CD8+ CD28 subsets. D) TNFα+ frequencies of CD4+ and CD8+ CD44+ T cell subsets. Lines connect data points from each individual dog. * = P<0.05; ** = P≤0.01; *** = P≤0.001; n = 6.

Article Snippet: For intracellular cytokine staining experiments, both mitogen-stimulated and control cells were concurrently incubated with Brefeldin A (B7651, Sigma-Aldrich, Saint Louis, MO, USA) at a dilution of 1 μl/ml in complete media during the 6-hour stimulation period. table ft1 table-wrap mode="anchored" t5 Table 1: caption a7 Antigen/ marker Conjugated fluorochrome Antigen species Antibody clone # Manufacturer Catalogue # Dilution Reference Cell Surface Viability dye Near infra-red N/A N/A Thermo Fisher {"type":"entrez-nucleotide","attrs":{"text":"L34975","term_id":"522218","term_text":"L34975"}} L34975 1:1000 N/A CD4 PE-Cy7 Canine YKIX302.9 eBioscience 25-5040-41 1:10 N/A CD4 PB Canine YKIX302.9 Biorad MCA1038PB 1:10 N/A CD8 PerCp-Cy5.5 Canine YCATE55.9 eBioscience 46-5080-41 1:25 N/A CD28 APC Canine 5B8 eBioscience 17-0282-41 1:10 N/A CD44 FITC Canine YKIX337.8 Thermo Fisher 11-5440-41 1:10 N/A CD62L PE Human FMC46 BioRad MCA1076PET 1:10 ( Bismarck et al., 2012 ; Hartley and Tarleton, 2015 ; Rothe et al., 2017 ; Schuberth et al., 2007 ) CD45RA Biotin Canine CA4.1D3 PFM Lab N/A 1:50 ( Cobbold and Metcalfe, 1994 ; Reis et al., 2005 ) Streptavadin BV711 N/A N/A Biolegend 405241 1:50 N/A CD25 PE Canine P4A10 eBioscience 12-0250-42 1:10 N/A Intracellular CD3 PB Human CD3-12 BioRad MCA1477PB 1:50 ( Monjazeb et al., 2016 ) CD3 FITC Human CD3-12 BioRad MCA1477F 1:50 ( Monjazeb et al., 2016 ) IFNγ PE Bovine CC302 BioRad MCA1783PE 1:10 ( Hartley and Tarleton, 2015 ; Pedersen et al., 2002 ) TNFα BV-785 Human MAB-11 Biolegend 502947 1:25 ( Moreira et al., 2015 ) Ki67 PE-Cy7 Human 20Raj1 eBioscience 25-5699-42 1:50 ( Galkowska et al., 1996 ) Open in a separate window APC, allophycocyanin; BV-711, brilliant violet™711; BV-785, brilliant violet™785; FITC, fluorescein; PB, pacific blue; PE, phycoerythrin; PE-Cy7, PE-cyanine 7; PE-TR, PE-texas red; PerCPCy5.5, peridinin chlorphyll protein-cyanine 5.5; PFM lab, Peter F. Moore laboratory; N/A, not applicable.

Techniques:

Journal: Developmental and comparative immunology

Article Title: Multi-color Flow Cytometry for Evaluating Age-Related Changes in Memory Lymphocyte Subsets in Dogs

doi: 10.1016/j.dci.2018.05.022

Figure Lengend Snippet: Alterations to T cell memory, CD28 and CD44 T cell subset frequencies in fresh PBMCs of young vs. aged dogs. A) Representative scatter plots illustrating memory subset frequencies of CD4+ and CD8+ T cells in a young/lean dog and an aged/lean dog. B) Frequencies of T cells with various memory phenotypes in young vs. aged dogs. C) Frequencies of CD28 subsets in young vs. aged dogs. D) Frequencies of CD44 subsets in young vs. aged dogs. Means and standard deviations are shown. ns = not significant; ** = P≤0.01; n = 6 per age group.

Article Snippet: For intracellular cytokine staining experiments, both mitogen-stimulated and control cells were concurrently incubated with Brefeldin A (B7651, Sigma-Aldrich, Saint Louis, MO, USA) at a dilution of 1 μl/ml in complete media during the 6-hour stimulation period. table ft1 table-wrap mode="anchored" t5 Table 1: caption a7 Antigen/ marker Conjugated fluorochrome Antigen species Antibody clone # Manufacturer Catalogue # Dilution Reference Cell Surface Viability dye Near infra-red N/A N/A Thermo Fisher {"type":"entrez-nucleotide","attrs":{"text":"L34975","term_id":"522218","term_text":"L34975"}} L34975 1:1000 N/A CD4 PE-Cy7 Canine YKIX302.9 eBioscience 25-5040-41 1:10 N/A CD4 PB Canine YKIX302.9 Biorad MCA1038PB 1:10 N/A CD8 PerCp-Cy5.5 Canine YCATE55.9 eBioscience 46-5080-41 1:25 N/A CD28 APC Canine 5B8 eBioscience 17-0282-41 1:10 N/A CD44 FITC Canine YKIX337.8 Thermo Fisher 11-5440-41 1:10 N/A CD62L PE Human FMC46 BioRad MCA1076PET 1:10 ( Bismarck et al., 2012 ; Hartley and Tarleton, 2015 ; Rothe et al., 2017 ; Schuberth et al., 2007 ) CD45RA Biotin Canine CA4.1D3 PFM Lab N/A 1:50 ( Cobbold and Metcalfe, 1994 ; Reis et al., 2005 ) Streptavadin BV711 N/A N/A Biolegend 405241 1:50 N/A CD25 PE Canine P4A10 eBioscience 12-0250-42 1:10 N/A Intracellular CD3 PB Human CD3-12 BioRad MCA1477PB 1:50 ( Monjazeb et al., 2016 ) CD3 FITC Human CD3-12 BioRad MCA1477F 1:50 ( Monjazeb et al., 2016 ) IFNγ PE Bovine CC302 BioRad MCA1783PE 1:10 ( Hartley and Tarleton, 2015 ; Pedersen et al., 2002 ) TNFα BV-785 Human MAB-11 Biolegend 502947 1:25 ( Moreira et al., 2015 ) Ki67 PE-Cy7 Human 20Raj1 eBioscience 25-5699-42 1:50 ( Galkowska et al., 1996 ) Open in a separate window APC, allophycocyanin; BV-711, brilliant violet™711; BV-785, brilliant violet™785; FITC, fluorescein; PB, pacific blue; PE, phycoerythrin; PE-Cy7, PE-cyanine 7; PE-TR, PE-texas red; PerCPCy5.5, peridinin chlorphyll protein-cyanine 5.5; PFM lab, Peter F. Moore laboratory; N/A, not applicable.

Techniques:

Journal: Developmental and comparative immunology

Article Title: Multi-color Flow Cytometry for Evaluating Age-Related Changes in Memory Lymphocyte Subsets in Dogs

doi: 10.1016/j.dci.2018.05.022

Figure Lengend Snippet: Cell surface and intracellular antibodies and markers used in flow cytometry studies

Article Snippet: For intracellular cytokine staining experiments, both mitogen-stimulated and control cells were concurrently incubated with Brefeldin A (B7651, Sigma-Aldrich, Saint Louis, MO, USA) at a dilution of 1 μl/ml in complete media during the 6-hour stimulation period. table ft1 table-wrap mode="anchored" t5 Table 1: caption a7 Antigen/ marker Conjugated fluorochrome Antigen species Antibody clone # Manufacturer Catalogue # Dilution Reference Cell Surface Viability dye Near infra-red N/A N/A Thermo Fisher {"type":"entrez-nucleotide","attrs":{"text":"L34975","term_id":"522218","term_text":"L34975"}} L34975 1:1000 N/A CD4 PE-Cy7 Canine YKIX302.9 eBioscience 25-5040-41 1:10 N/A CD4 PB Canine YKIX302.9 Biorad MCA1038PB 1:10 N/A CD8 PerCp-Cy5.5 Canine YCATE55.9 eBioscience 46-5080-41 1:25 N/A CD28 APC Canine 5B8 eBioscience 17-0282-41 1:10 N/A CD44 FITC Canine YKIX337.8 Thermo Fisher 11-5440-41 1:10 N/A CD62L PE Human FMC46 BioRad MCA1076PET 1:10 ( Bismarck et al., 2012 ; Hartley and Tarleton, 2015 ; Rothe et al., 2017 ; Schuberth et al., 2007 ) CD45RA Biotin Canine CA4.1D3 PFM Lab N/A 1:50 ( Cobbold and Metcalfe, 1994 ; Reis et al., 2005 ) Streptavadin BV711 N/A N/A Biolegend 405241 1:50 N/A CD25 PE Canine P4A10 eBioscience 12-0250-42 1:10 N/A Intracellular CD3 PB Human CD3-12 BioRad MCA1477PB 1:50 ( Monjazeb et al., 2016 ) CD3 FITC Human CD3-12 BioRad MCA1477F 1:50 ( Monjazeb et al., 2016 ) IFNγ PE Bovine CC302 BioRad MCA1783PE 1:10 ( Hartley and Tarleton, 2015 ; Pedersen et al., 2002 ) TNFα BV-785 Human MAB-11 Biolegend 502947 1:25 ( Moreira et al., 2015 ) Ki67 PE-Cy7 Human 20Raj1 eBioscience 25-5699-42 1:50 ( Galkowska et al., 1996 ) Open in a separate window APC, allophycocyanin; BV-711, brilliant violet™711; BV-785, brilliant violet™785; FITC, fluorescein; PB, pacific blue; PE, phycoerythrin; PE-Cy7, PE-cyanine 7; PE-TR, PE-texas red; PerCPCy5.5, peridinin chlorphyll protein-cyanine 5.5; PFM lab, Peter F. Moore laboratory; N/A, not applicable.

Techniques: Flow Cytometry, Marker