A fraction of enriched PBMCs and enriched tumor-infiltrating lymphocytes (TIL) were plated in a 96 well U-bottom plate and stimulated with Dynabeads Human T-Activator CD3/CD28 for T cell Expansion and Activation (Thermo Fisher Scientific, Cat #11132D) at a ratio of 1 bead:1 cell for 12 h in cMedia in the presence of protein transport inhibitor cocktail 500X (Thermo Fisher Scientific, Cat #00-4980-03). The samples were washed with 1X PBS, and underwent staining with Fixable Viability Dye (eFluor 780 Thermo Fisher Scientific, Cat # 65-0865-14), followed by fluorophore conjugated antibodies specific for human CD4 (BV570 Biolegend, Cat 300534), CD8 (BV650 Biolegend, Cat # 301041), PD-1 (PE-Cy7 Biolegend, Cat # 329918) and CD3 (AF700 BD Biosciences, Cat # 557943). The samples then underwent fixation and permeabilization (eBioscience, Cat # 88-8824-00), after which they stained by fluorophore conjugated antibodies specific for human FoxP3 (Pac Blue BD Biosciences, Cat # 560460), IFNγ (FITC BD Biosciences, Cat # 554700), IL-2 (PE Thermo Fisher Scientific, Cat # 562462) and TNFα (APC Biolegend, Cat # 502913). Manufacturer specified isotype control antibodies were used to determine positive signal and gating strategies. Data were analyzed using FlowJo Software (Tree Star, Mac version 9.9.4).
Pac blue
Manufactured by BD
Pac Blue is a fluorescent dye used in flow cytometry and microscopy applications. It is designed to label and detect specific biological targets, such as cells or molecules, for analysis. Pac Blue exhibits excitation and emission spectra suitable for common fluorescence detection systems.
Automatically generated - may contain errors
Lab products found in correlation
Af700, by BD (1 mentions)
Dynabeads human t activator cd3 cd28 for t cell expansion and activation, by Thermo Fisher Scientific (1 mentions)
Bv570, by BioLegend (1 mentions)
Fixable viability dye efluor 780, by Thermo Fisher Scientific (1 mentions)
Protein transport inhibitor cocktail 500x, by Thermo Fisher Scientific (1 mentions)
Pe cy7, by BioLegend (1 mentions)
Efluor 780, by Thermo Fisher Scientific (1 mentions)
Pe cy7, by BD (1 mentions)
Percp cy5, by BioLegend (1 mentions)
Pe texas red, by BD (1 mentions)
3 protocols using pac blue
1
Multiparametric Profiling of Activated T Cells
2
Comprehensive Immune Profiling of PBMCs and TILs
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A fraction of enriched PBMCs and enriched TIL were plated in a 96 well U-bottom plate. The samples were washed with 1X PBS and underwent staining with Fixable Viability Dye (eFluor 780 Thermo Fisher Scientific, Cat # 65-0865-14), followed by fluorophore conjugated antibodies specific for human CD45RO (BV570 Biolegend, Cat # 304226), CD8 (BV650 Biolegend, Cat # 301041), LAG-3 (FITC BMS), TIM-3 (PE Biolegend, Cat # 345005), CD4 (PE-Texas Red BD Biosciences, Cat # 562316), CD45RA (PerCP-Cy5.5 Biolegend, Cat # 304122), PD-1 (PE-Cy7 BD Biosciences, Cat # 561272) and CD3 (AF700 BD Biosciences, Cat # 557943). The samples then underwent fixation and permeabilization (eBioscience, Cat # 88-8824-00), after which they stained by fluorophore conjugated antibodies specific for human FoxP3 (Pac Blue BD Biosciences, Cat # 560460) and CTLA-4 (APC BD Biosciences, Cat # 560938). Manufacturer specified isotype control antibodies were used to determine positive signal and gating strategies. Data were analyzed using FlowJo Software (Tree Star, Mac version 9.9.4).
3
Measuring DC Activation by Tregs
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This assay has been previously described (16 ). Spleens were collected from adult, sex-matched NOD/ShiLtJ or NOD-HLA-A2 mice and enzymatically digested (STEMCELL Technologies). CD11c+ DCs were positively magnetically isolated (STEMCELL Technologies). DCs were plated at a 1:2 ratio with Tregs and left to co-incubate for 2 days, at which point CD11c+ (SB780, clone N418, BD Biosciences) DC expression of CD80 (BUV395, clone 16–10A1, BD Biosciences), CD86 (BV650, clone GL1, BD Biosciences) and I-Ag7 (PacBlue or AF488, clone 10.2–16, provided by Dr. Brian Fife at the University of Minnesota) were measured by flow cytometry.
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