RPMI1640 medium containing 10% FCS was used for CD4 naïve T cell cultures. The following antibodies were purchased from BD BioSciences (San Jose, CA): anti-mouse CD3 (145-2C11), anti-mouse CD28 (37.51), anti-mouse IL-2, anti-CD4-PerCP, anti-CD4-PECy7, anti-CD8-FITC, anti-CD25-APC, anti-CD25-PE, anti-CD44-FITC, anti-CD62L-PE, anti-BrdU-FITC. Anti-FoxP3-APC, anti-IFNγ-APC, and anti-IL-17A-PE were from eBioscience (San Diego, CA). Anti-Helios, anti-CD122 and anti-GITR were from Biolegend (San Diego, CA). Recombinant mouse IL-2 and IL-7 were purchased from R&D Systems (Minneapolis, MN) and ovalbumin was from Sigma-Aldrich (St Louis, MO).
Anti gitr
Manufactured by BioLegend
Sourced in United States
Anti-GITR is a monoclonal antibody targeting the GITR (Glucocorticoid-Induced TNFR-Related Protein) receptor. GITR is a co-stimulatory molecule expressed on T cells, natural killer cells, and regulatory T cells. The Anti-GITR antibody can be used in applications such as flow cytometry, immunohistochemistry, and in vitro cell stimulation.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd25 pe, by BD (2 mentions)
Anti helios, by BioLegend (2 mentions)
Anti cd3, by BioLegend (2 mentions)
Anti icos, by BioLegend (2 mentions)
Anti cd4, by BioLegend (2 mentions)
Anti foxp3 apc, by Thermo Fisher Scientific (1 mentions)
Anti cd8 fitc, by BD (1 mentions)
Anti brdu fitc, by BD (1 mentions)
Anti cd4 percp, by BD (1 mentions)
Anti cd44 fitc, by BD (1 mentions)
Anti cd4 pe cy7, by BD (1 mentions)
Anti il 17a pe, by Thermo Fisher Scientific (1 mentions)
Anti cd62l pe, by BD (1 mentions)
Anti cd25 apc, by BD (1 mentions)
Ovalbumin (ova), by Merck Group (1 mentions)
Anti ifn γ apc, by Thermo Fisher Scientific (1 mentions)
Anti mouse il 2, by BD (1 mentions)
Anti cd122, by BioLegend (1 mentions)
Anti cd28, by BioLegend (1 mentions)
Tgf β, by Thermo Fisher Scientific (1 mentions)
4 protocols using anti gitr
1
CD4+ Naïve T Cell Culture Protocol
2
Regulatory T Cell Isolation and Characterization
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Tregs were prepared according to the all-trans retinal method as described previously
[17 (link)]. Briefly, spleens were harvested from
C57BL/6 mice. CD4+ T cells were isolated by using a Dynal® CD4 Negative
Isolation Kit (Dynal Biotect, Bromborough, Wirral, UK). These cells were then cultured
with anti-CD3 (BioLegend, San Diego, CA, USA), anti-CD28 (BioLegend, San Diego, CA, USA),
TGF-β (PeproTech, London, UK), and retinal acid (Sigma-Aldrich, St. Louis, MO, USA) for 72
h. CD4+ CD25+ cells were sorted out of the culture by flow cytometry
and analyzed with cell surface antibodies, such as anti-CD4 -FITC (Abcam, Cambridge, UK),
anti-CD25-PE (BD PharMingen, San Diego, CA, USA), anti-FOXP3-APC (BD PharMingen, San
Diego, CA, USA), anti-CD44 (BioLegend, San Diego, CA, USA), anti-GITR (BioLegend, San
Diego, CA, USA), and anti-CTL4 (BioLegend, San Diego, CA, USA).
[17 (link)]. Briefly, spleens were harvested from
C57BL/6 mice. CD4+ T cells were isolated by using a Dynal® CD4 Negative
Isolation Kit (Dynal Biotect, Bromborough, Wirral, UK). These cells were then cultured
with anti-CD3 (BioLegend, San Diego, CA, USA), anti-CD28 (BioLegend, San Diego, CA, USA),
TGF-β (PeproTech, London, UK), and retinal acid (Sigma-Aldrich, St. Louis, MO, USA) for 72
h. CD4+ CD25+ cells were sorted out of the culture by flow cytometry
and analyzed with cell surface antibodies, such as anti-CD4 -FITC (Abcam, Cambridge, UK),
anti-CD25-PE (BD PharMingen, San Diego, CA, USA), anti-FOXP3-APC (BD PharMingen, San
Diego, CA, USA), anti-CD44 (BioLegend, San Diego, CA, USA), anti-GITR (BioLegend, San
Diego, CA, USA), and anti-CTL4 (BioLegend, San Diego, CA, USA).
3
Immunophenotyping and T Cell Function
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ASTRLs and PBMCs were immunophenotyped for various cell surface markers by flow cytometry with fluorophore conjugated human anti-CD3, anti-CD4, anti-CD8, anti-CD25, anti-CD127, anti-CD39 and anti-CD73 anti-CTLA4, anti-GITR, anti-ICOS, anti-CD45RA, anti-CD226, anti-LAP, anti-GARP, anti-CD56, anti-CD16, anti-CD19, anti-CD11b, anti-CD38, anti-CD27, and anti-CD24 (Biolegend). The data were acquired using a Canto II cytometer (BD Biosciences) and analyzed using Flowjo. The gating strategy for phenotyping included initial gating of a live PBMC population followed by the CD3+CD4+ population. The expression of CD25, CD127, CD39, and CD73 were expressed as % of the CD3+CD4+ population.
T cell proliferation and suppression was determined by CFSE dye dilution of the responder cells. Analysis of CFSE distribution was performed on Flowjo Proliferation platform and data are represented by Replication Index (RI). RI, determines the fold-expansion of only the responding cells, and it is the average number of divisions that all cells have undergone after they had been stained by a cell proliferation dye (13 (link)). The percentage of suppression was calculated from proliferation and suppression values.
T cell proliferation and suppression was determined by CFSE dye dilution of the responder cells. Analysis of CFSE distribution was performed on Flowjo Proliferation platform and data are represented by Replication Index (RI). RI, determines the fold-expansion of only the responding cells, and it is the average number of divisions that all cells have undergone after they had been stained by a cell proliferation dye (13 (link)). The percentage of suppression was calculated from proliferation and suppression values.
4
Comprehensive Murine Regulatory T Cell Analysis
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For flow cytometry, the cell surface was stained with the following antibodies specific for mouse proteins: anti-CD4, anti-CCR6, anti-CD25, anti-CTLA-4, anti-GITR, and anti-ICOS (BioLegend). Mouse Regulatory T Cell Staining Kit (eBioscience; San Diego, CA) was used to stain the transcription factors with anti-Foxp3 (Biolegend) and anti-RORγt antibodies (BD), anti-Blimp-1 (Biolegend) and anti-Helios (Biolegend), or intracellular cytokine staining with anti-IL-17A and anti-IL-10 (BioLegend). Data were acquired using the FACSVerseFlow Cytometer (Becton Dickinson; Mountain View, CA) or the SH800 Cell Sorter (Sony, Tokyo, Japan) and analyzed with FlowJo software (Tree Star; Ashland, OR).
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