Lipopolysaccharides (0111:B4) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Antibodies specific for GST (sc-459) and β-actin (sc-47778) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies specific for GRP78 (ab32618), CD14 (ab182032), and TLR4 (ab22048) were obtained from Abcam (Cambridge, UK). Antibodies specific for interferon regulatory transcription factor (IRF)3 (4302) and IRF3 phosphorylated at Ser396(4947) were obtained from Cell Signaling Technology (Danvers, MA, USA). F(ab′)2-goat anti-mouse Alexa Fluor®555 (AF555) was purchased from Thermo Fisher Scientific. Anti-TLR4-PE was obtained from PharMingen (Becton Dickinson, Franklin Lakes, NJ, USA). Anti-LAMP1-PE, anti-CD14-PECy7, and permeabilization buffer were obtained from eBioscience (San Diego, CA, USA).
Anti tlr4 pe
Manufactured by BD
Sourced in United States
Anti-TLR4-PE is a fluorescently labeled antibody that specifically binds to the Toll-like receptor 4 (TLR4) on the surface of cells. It can be used for the detection and analysis of TLR4-expressing cells in various applications, such as flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Permeabilization buffer, by Thermo Fisher Scientific (1 mentions)
Ab182032, by Abcam (1 mentions)
Ab22048, by Abcam (1 mentions)
Ab32618, by Abcam (1 mentions)
β actin sc 47778, by Santa Cruz Biotechnology (1 mentions)
Sc 459, by Santa Cruz Biotechnology (1 mentions)
Anti cd14 pecy7, by Thermo Fisher Scientific (1 mentions)
Facscalibur, by BD (1 mentions)
Trypsin edta, by Merck Group (1 mentions)
Annexin 5 fitc, by BD (1 mentions)
Anti cd3 percp, by BD (1 mentions)
Anti cd56 fitc, by BD (1 mentions)
Anti nkg2d pe, by BD (1 mentions)
Anti cd16 fitc, by Miltenyi Biotec (1 mentions)
Anti nkp44 pe, by BioLegend (1 mentions)
Anti 2b4 fitc, by BioLegend (1 mentions)
Anti nkp30 pe, by BioLegend (1 mentions)
Anti tlr 2 pe, by BD (1 mentions)
Anti nkp46 pe, by BD (1 mentions)
Anti cd56 apc, by BD (1 mentions)
5 protocols using anti tlr4 pe
1
Lipopolysaccharide-Induced Signaling Pathway Analysis
2
Characterization of Isolated NK Cells
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The purity of isolated NK cells (>95%) and expression of surface receptors were determined by flow cytometry using a FACSCalibur (BD-Biosciences). NK cell population was defined as NKp46+CD3− cells.
Changes in the expression of NK cell receptors were examined using the following antibodies: anti-NKp30 PE (Biolegend), anti-NKp44 PE (Biolegend), anti-NKG2D PE (BD-Biosciences), anti-CD16 FITC (Miltenyi Biotec), anti-CD56 FITC and APC (BD-Biosciences), anti-2B4 FITC (Biolegend), anti-NTB-A PE (Biolegend), anti-Dectin-1 PE, anti-TLR-2 PE and anti-TLR-4 PE (BD-Biosciences). Apoptotic NK cells were assessed after an incubation time of 9 h using Annexin V FITC (BD-Biosciences) after staining cells with the surface marker antibodies anti-NKp46 PE, anti-CD3 PerCP and anti-CD56 APC. Intracellular expression of CD56 after incubation with germ tubes for 6 h was investigated by firstly staining CD56 on the cell surface with anti-CD56 FITC (BD-Biosciences). Then, cells were fixed (4% formaldehyde), permeabilized (Wash Perm, BD-Biosciences) and intracellular CD56 was stained using anti-CD56 APC (BD-Biosciences). To remove surface markers, NK cells were incubated in 0.5% trypsin-EDTA (Sigma) for 30 min at 37 °C before samples were stained. All data were analyzed with FlowJo software (Tree Star Inc.).
Changes in the expression of NK cell receptors were examined using the following antibodies: anti-NKp30 PE (Biolegend), anti-NKp44 PE (Biolegend), anti-NKG2D PE (BD-Biosciences), anti-CD16 FITC (Miltenyi Biotec), anti-CD56 FITC and APC (BD-Biosciences), anti-2B4 FITC (Biolegend), anti-NTB-A PE (Biolegend), anti-Dectin-1 PE, anti-TLR-2 PE and anti-TLR-4 PE (BD-Biosciences). Apoptotic NK cells were assessed after an incubation time of 9 h using Annexin V FITC (BD-Biosciences) after staining cells with the surface marker antibodies anti-NKp46 PE, anti-CD3 PerCP and anti-CD56 APC. Intracellular expression of CD56 after incubation with germ tubes for 6 h was investigated by firstly staining CD56 on the cell surface with anti-CD56 FITC (BD-Biosciences). Then, cells were fixed (4% formaldehyde), permeabilized (Wash Perm, BD-Biosciences) and intracellular CD56 was stained using anti-CD56 APC (BD-Biosciences). To remove surface markers, NK cells were incubated in 0.5% trypsin-EDTA (Sigma) for 30 min at 37 °C before samples were stained. All data were analyzed with FlowJo software (Tree Star Inc.).
3
Profiling Parasite-Induced Innate Immune Responses
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PBMCs were either infected with parasites of L braziliensis or left uninfected. Infected and uninfected PBMCs were then incubated separately for 8 h and 24 h at 37°C, 5% CO2. Cells were then stained with anti-CD14 monoclonal antibodies (PerCP Cy-5.5), anti-CD16 (APC), anti-TLR2 (PE) and anti-TLR4 (PE) for 15 min 4°C in the dark (BD-Bioscience). The cells were washed with PBS (1,500 rpm, 5 min, 4°C), fixed with 4% paraformaldehyde, and permeabilized with Perm Wash solution for 15 min at 4°C in the dark (BD-Bioscience). Intracellular staining was performed with anti-TNF and anti-IL-10 (FITC) antibodies for 30 min. After this period, the cells were washed and suspended in 400 μl PBS for flow cytometry analysis on BD FACS CANTOII. A total of 200,000 events were acquired. Data analysis was performed using FlowJo program (Free Star Inc.).
4
Flow Cytometry Analysis of Macrophage Markers
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The expression of surface TLR4, CD11c and CCR2 on each macrophage was measured by flow cytometric analysis. Briefly, the cells (2 × 105 cells) were treated with FcR blocker (mouse normal serum, Biomeda, Foster City, CA, USA) for 30 min on ice, and then stained in ice-cold PBS containing 0.3% BSA and 0.05% NaN3 with the following antibodies: Anti-TLR4-PE, anti-CD11c-FITC and isotype controls (BD Bioscience, San Diego, CA, USA), and CCR2 (anti-mouse CCR2 rabbit polyclonal antibody, Abcam, Cambridge, UK). F-actin was stained by phalloidin (Acti-stain™ 555 phalloidin, Cytoskeleton, Denver, CO, USA). The flow cytometric analysis was performed using the FACSCalliber™ Flow Cytometry System with the CellQuest software package (BD, Franklin Lakes, NJ, USA). The change in the mean fluorescence intensity (MFI) between anti-TLR4 and CD11c antibodies and each isotype control were obtained to quantify the expression of each marker.
5
Macrophage NF-κB Activation Modulation
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Peritoneal macrophages isolated from male C57BL6/j mice (8-week-old) were treated with 1 µg of LPS (Alexa Fluor 488, E. coli Serotype O55:B5, Invitrogen Molecular Probes, Eugene, Oregon, USA) in the absence or presence of various concentrations of recombinant human CETP (Roar Biomedical, NY, USA) (0.1, 0.5, and 1.0 µg/mL) diluted in RPMI and stained with the monoclonal antibodies; we utilized anti-mouse F4/80 PerCP/Cy5.5 a monoclonal antibody directed specifically against the mouse macrophage; for TLR4 we utilized anti-TLR4-PE (BD Biosciences, Franklin Lakes, NJ, USA). NF-κB: rabbit anti-mouse Phospho-NF-κB p65 Alexa Fluor 647 Conjugate (Cell Signaling Technology, Inc., Boston, MA, USA) was diluted at 1 : 100. We utilized Cytofix (BD Biosciences, San Jose, USA) for surface label and Cytofix/Cytoperm (BD Biosciences, San Jose, USA) for intracellular label. Basal fluorescence was determined in unmarked cells and compensation made with cells labeled with the respective fluorochromes. Samples were acquired on FACSCanto, using FACSDiva software (BD Biosciences) and then were analyzed with FlowJo software (Tree Star, Ashland, OR, USA). Fluorescence voltages were determined using matched unstained cells. Fifty thousand events were acquired in a live mononuclear gate.
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