cd4 rat igg2a ykix302 9 fitc bio rad  (Bio-Rad)

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 violetTM711; BV-785, brilliant violetTM785; 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 CD4+ and CD8+ T cells in young versus aged dogs. A) Frequencies of IFNγ+ and TNFα+ CD4+ and CD8+ T cells in young and aged dogs after a 6 hour ConA stimulation. B) Proliferative fraction of CD4+ and CD8+ T cells in young and aged dogs, determined by Ki67 expression after 2 days of mitogen stimulation. Means and standard deviations are shown. * = P<0.05; ** = P≤0.01; n = 4–6 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 violetTM711; BV-785, brilliant violetTM785; 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: Expressing

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 violetTM711; BV-785, brilliant violetTM785; 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: Changes in frequencies of canine memory T cell subsets between mitogen-stimulated and unstimulated PBMCs. A) Representative scatter plot illustrating the frequencies of CD4+ and CD8+ T cells with various memory phenotypes with and without 6 hour ConA stimulation. The example shown is from an aged, overweight male beagle. B) Summary of changes to the frequencies of CD4+ and CD8+ T cells with naïve- and TEMRA-like phenotypes after mitogen stimulation in young and aged dogs. Control PBMCs were incubated in complete media concurrently with stimulated PBMCs. Lines connect data points from each individual dog. * = P<0.05; 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 violetTM711; BV-785, brilliant violetTM785; 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: Control, Incubation

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 violetTM711; BV-785, brilliant violetTM785; 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

Journal: Frontiers in immunology

Article Title: Comparison of Phenotypic and Functional Characteristics Between Canine Non-B, Non-T Natural Killer Lymphocytes and CD3 + CD5 dim CD21 - Cytotoxic Large Granular Lymphocytes.

doi: 10.3389/fimmu.2018.00841

Figure Lengend Snippet: Figure 3 | Phenotypic changes of purified CD3+CD5dimCD21− cells during culture for 21 days. (A) CD3+CD5dimCD21− cells were purified from freshly isolated PBMCs using a cell sorter. The purity of CD3+CD5dimCD21− cells after sorting was more than 98% (n = 5). (B) Phenotypic analysis of the purified CD3+CD5dimCD21− cells during culture for 21 days. CD3−CD5−CD21− cells were proliferated from purified CD3+CD5dimCD21− cells, and the majority of expanded cells in culture were CD3−CD5−CD21− after 21 days. The phenotype of most of these expanded CD3−CD5−CD21− cells was CD4− CD8+/− after 21 days of stimulation. The results shown are representative results from one of five different donors. (C) The rate of proliferation was calculated by counting the total number of viable cells in culture. Values represent the mean ± SD (n = 5).

Article Snippet: Based on these results, the interrelationship between these two cell populations as putative canine NK cells was assumed, and we hypothesized that phenotypic modulation might occur between TaBle 1 | Antibodies used for flow cytometry in this study. antibody clone conjugates supplier Primary antibody (isotype) Mouse anti Human CD14 (IgG2a, κ)* M5E2 PE-Cy7 BD Pharmingen Mouse anti-Canine CD21 (IgG1) CA2.1D6 RPE Bio-Rad Mouse anti-Dog CD3 (IgG1) CA17.2A12 FITC Bio-Rad Rat anti-Dog CD5 (IgG2a) YKIX322.3 APC Bio-Rad Rat anti-Dog CD4 (IgG2a) YKIX302.9 RPE Bio-Rad Rat anti-Dog CD8 (IgG1) YCATE55.9 RPE Bio-Rad Mouse anti-Canine CD21 (IgG1) CA2.1D6 – Bio-Rad Mouse anti-Dog CD3 (IgG1) CA17.2A12 – Bio-Rad Mouse anti-Dog CD11c (IgG1) CA11.6A1 – Bio-Rad Mouse anti-Dog CD11d (IgG1) CA11.8H2 – Bio-Rad Canine TCRαβ (IgG1) CA15.8G7 – Perter Moorea Canine TCRγδ (IgG2a) CA20.8H1 – Perter Moorea intracellular staining Mouse anti Human Ki-67 (IgG1, kappa)* 20Raj1 PE-Cyanine7 Invitrogen Mouse anti Human Granzyme B (IgG1, κ)** GB11 PE BD Pharmingen Mouse anti Human EOMES (IgG1, kappa)b WD1928 PE-Cyanine7 Invitrogen Mouse anti Human/Mouse T-bet (IgG1, kappa)*** eBio4B10 (4B10) PE Invitrogen isotype control Mouse IgG1 Negative Control – FITC Bio-Rad Mouse IgG1 kappa Isotype Control P3.6.2.8.1 PE-Cyanine7 Invitrogen Mouse IgG1 kappa Isotype Control P3.6.2.8.1 PE Invitrogen Mouse IgG1, κ Isotype Control MOPC-21 PE BD Pharmingen secondary antibody Goat anti-Mouse IgG (H + L) Secondary Antibody – Pacific Blue Invitrogen Cross reactivity to the canine equivalent reported* by the supplier.

Techniques: Purification, Isolation