The following directly conjugated antibodies were used: CD3-APCH7, CD27-Alexa Fluor700, MIP1β-PE, CTLA4-APC, TNFα-Cy7PE, IL2-Cy55PCP (BD Biosciences); CD4-Cy55PE, Streptavidin (SA)-quantum dot (QD) 655, Aqua amine reactive dye (Invitrogen); CD45RO-TRPE, 2B4-Cy5PE (Beckman Coulter); IFNγ-eFluor450 (eBioscience); Biotinylated PD-1 (clone BAF1086; R&D Systems); CD8-BV711 (Biolegend). The following antibodies were conjugated in our laboratory: CD8-QD800 and CD57-QD565.
Mip1β pe
Manufactured by BD
Sourced in United States
MIP1β-PE is a fluorescent-labeled antibody that detects human MIP1β protein. It is used in flow cytometry applications to identify and quantify cells expressing MIP1β.
Automatically generated - may contain errors
Lab products found in correlation
Cd56 pe cy7, by BD (2 mentions)
Ifn γ apc, by BD (2 mentions)
Cd16 apc cy7, by BD (2 mentions)
Golgistop, by BD (2 mentions)
Brefeldin a, by Merck Group (2 mentions)
Cd3 apc h7, by BD (1 mentions)
Cd8 bv711, by BioLegend (1 mentions)
Ifn γ efluor450, by Thermo Fisher Scientific (1 mentions)
Ctla4 apc, by BD (1 mentions)
Aqua amine reactive dye, by Thermo Fisher Scientific (1 mentions)
Cd27 alexa fluor700, by BD (1 mentions)
Cd4 cy55pe, by Thermo Fisher Scientific (1 mentions)
Facscanto 2, by BD (1 mentions)
Ifn γ pe cy7, by BD (1 mentions)
Golgistop solution, by BD (1 mentions)
Cd8 percp, by BioLegend (1 mentions)
Cd8 percp, by BD (1 mentions)
Sodium azide, by Merck Group (1 mentions)
Il 2 apc, by BD (1 mentions)
Tnf α, by BioLegend (1 mentions)
5 protocols using mip1β pe
1
Multiparameter Flow Cytometry Panel
2
Flow Cytometric Analysis of HLA Tetramers and Intracellular Cytokines
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All flow cytometric measurements were performed on a FACSCanto II (BD) and evaluated using FlowJo version 9.2 (Tree Star, Ashland, OR, USA). For HLA tetramer analysis, T cells were washed in FACS buffer containing 2% FCS, 2 mM EDTA (Carl Roth, 8040) and 0.02% sodium azide (Merck Millipore, 8223350100) in PBS, followed by a staining for dead cells using Live/Dead fixable Aqua dead cell stain kit (Invitrogen, L34957). Afterwards, cells were washed and incubated with 2.5 µg/mL of the respective tetramer in tetramer solution (FACS buffer with 50% FCS) for 30 min at RT. Finally, cell surface molecules were stained with CD8-PerCP (Biolegend, 301030) for 20 min at 4°C.
Intracellular cytokine staining was performed by incubating in vitro primed T cells with 10 µg/mL candidate or control peptide, Golgi-Stop-Solution (BD Bioscience, 554724) and anti-CD107a-FITC antibody (BD Bioscience, 555800) for 6 h. Afterwards, cells were stained for dead cells using Live/Dead fixable Aqua dead cell stain kit followed by staining with CD8-PerCP for 20 min. Intracellular staining was performed after 30 min permeabilization with Cytoperm/Cytofix solution (BD Bioscience, 554722) using the following antibodies: IFNγ-PE-Cy7, IL-2-APC, TNFα-PacificBlue and MIP-1β-PE (all BD Bioscience, 557844, 554567 and 550078; except TNFα, Biolegend, 502920).
Intracellular cytokine staining was performed by incubating in vitro primed T cells with 10 µg/mL candidate or control peptide, Golgi-Stop-Solution (BD Bioscience, 554724) and anti-CD107a-FITC antibody (BD Bioscience, 555800) for 6 h. Afterwards, cells were stained for dead cells using Live/Dead fixable Aqua dead cell stain kit followed by staining with CD8-PerCP for 20 min. Intracellular staining was performed after 30 min permeabilization with Cytoperm/Cytofix solution (BD Bioscience, 554722) using the following antibodies: IFNγ-PE-Cy7, IL-2-APC, TNFα-PacificBlue and MIP-1β-PE (all BD Bioscience, 557844, 554567 and 550078; except TNFα, Biolegend, 502920).
3
Phenotyping NK Cell Activation
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Enzyme-linked immunosorbent assay plates were coated with measles antigen and incubated with samples diluted 1:20, or PBS as negative control, for 2 hours at 37°C. Natural killer cells were isolated from buffy coats collected from blood bank donors. CD107a-PE/Cy5 (BD), brefeldin A (Sigma), GolgiStop (BD), and NK cells were added to the antigen-coated plate and incubated for 5 hours at 37°C. Next, cells were transferred to a V-bottom plate and stained with CD56-PE/Cy7, CD16-APC/Cy7, and CD3-AlexaFluor700 (all BD) for 15 minutes at room temperature. Cells were washed and fixed in Fixation Medium A (Life Technologies) before intracellular staining with MIP-1β-PE and IFN-γ-APC (BD) in Permeabilization Medium B (Life Technologies) for 15 minutes at room temperature. The percentages of positive NK cells for CD107a, IFN-γ, and MIP-1β were determined by flow cytometry.
4
NK Cell Cytotoxicity and Cytokine Profiling
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NK cell responses to target cell lines were determined as previously described [13] (link). Briefly, mononuclear cells were incubated with MHC class I-devoid K562, 721.221, or antibody-coated p815 cell lines (ATCC) at an effector to target ratio of 10∶1. CD107a-PECy5 (10 µl/ml), brefeldin A (0.5 µg/ml, Sigma-Aldrich) and monensin (0.5 µg/ml, GolgiStop; BD Biosciences) were added at the start of the incubation. After five hours, cells were washed and stained to identify NK cells as above. Cells were fixed, permeabilized and stained for intracellular cytokines using MIP1β-PE and IFNγ-FITC (both BD Biosciences) antibodies. Multi-parameter flow cytometry was performed on a 4 laser BD LSRII system and analyzed as above.
5
Activated NK Cell Effector Functions Assay
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Intracellular staining was used to assess the effector functions of activated natural killer (NK) cells isolated by negative selection (RosetteSep kit, Stemcell technologies) from fresh whole blood from healthy donors, as previously described (24 (link)), with the following modifications – 50 μl of 0.25 mg/ml IgG purified from plasma (data not shown) or 50 μl CVL were incubated with the one of four HIV antigens Con6 gp120/B (Consensus Group M gp120), gp41 (Ectodomain) (HIV-1), p66 RT, and p24 (HIV-1/Clade B/C CN54) (Immune Technology, California, USA or ImmunoDX, Massachusetts, USA) and 50,000 NK cells/well. Following incubation with monensin and Brefeldin A as well as CD107a, cells were stained with CD3− A700, CD56 PE-Cy-7, CD16 APC-Cy7, IFN-γ APC, and MIP1-β PE (BD Biosciences, California, USA) and were then fixed with BD cell fix and then acquired on a LSR Fortessa (BD Biosciences, California, USA). Enriched NK cells were acquired and selected for singlets, followed by CD3 negative lymphocytes that were CD56+/CD16+ (Figure 2 ). The release of CD107a, IFN-γ and MIP-1β was used to assess frequency of activated NK cells to determine NK-cell activated ADCC (Figure 2 ) (9 (link)). When calculating HIV-specific antiviral activities, the non-specific ADCC activity obtained as background activity from PBS/NHS was subtracted from HIV-specific antibody-mediated ADCC activities.
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