Approximately 5.0 × 105 cells were pelleted and resuspended in 100 μL staining buffer (PBS, 0.5% bovine serum albumin, 2 mM EDTA). Cells were stained with a 50 μL cocktail containing staining buffer, HLA‐DR‐PE‐Cy7, CCR2‐AmCyan, CX3CR1‐PerCp‐Cy5.5, TLR‐4‐PE, and TLR‐2‐FITC (Biolegend) for 30 mins at room temperature, and washed twice in staining buffer. Cells were resuspended in staining buffer and analyzed using a Beckman Coulter Gallios flow cytometer. Fluorescence is presented as the natural log mean fluorescent intensity (MFI).
Tlr 4 pe
Manufactured by BioLegend
Sourced in United States
TLR-4-PE is a flow cytometry reagent that binds to Toll-like receptor 4 (TLR4) on the surface of cells. It is conjugated with the fluorescent dye phycoerythrin (PE), allowing for the detection and quantification of TLR4-expressing cells using flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Canto 2, by BD (2 mentions)
Cx3cr1 percp cy5, by BioLegend (1 mentions)
Tlr2 fitc, by BioLegend (1 mentions)
Hla dr pe cy7, by BioLegend (1 mentions)
Gallios flow cytometer, by Beckman Coulter (1 mentions)
Cd43 fitc, by BioLegend (1 mentions)
Ceacam1 pe, by R&D Systems (1 mentions)
Cxcr2 fitc, by BioLegend (1 mentions)
Cd64 apc, by BioLegend (1 mentions)
Cd62l pe, by BioLegend (1 mentions)
Cd11b pe, by BioLegend (1 mentions)
Nf κb activation nuclear translocation assay kit, by Beyotime (1 mentions)
Cd27 apc, by BioLegend (1 mentions)
Cxcr4 apc, by BioLegend (1 mentions)
Cd138 pe, by BioLegend (1 mentions)
Cd38 fitc, by BioLegend (1 mentions)
4 protocols using tlr 4 pe
1
Multiparametric Flow Cytometry Analysis
2
Multicolor Flow Cytometry Immune Cell Analysis
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After the erythrocytes were lysed, whole blood samples were stained using TLR1-PE, TLR2-AF647, TLR4-PE, CXCR1-PE, CXCR2-FITC, FPR1-APC, CD62L-PE, CD43-FITC, CD11b-PE, CD64-APC (Biolegend, USA), CEACAM-1-PE (R&D, USA), and CD16-BV510 (Sony, USA) antibodies and then analyzed by flow cytometry (Canto-II, BD, USA). Isotype-matched immunoglobulins were used as negative controls.
3
Assessing NF-κB and ROS in Macrophages
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The effects of extract and MgCl2 on NF-κB activity were evaluated with an NF-κB activation-nuclear translocation assay kit (Beyotime, China). THP-1 cell-derived macrophages were seeded into six-well plates with extract and MgCl2 for 1 h and then added with or without LPS for 30 min. After rinsing, fixation, and blocking, macrophages were incubated with p-65 primary antibody at 4°C overnight. Cells were subsequently incubated with cy3-conjugated secondary antibody for 1 h and then stained with DAPI for 5 min at room temperature. Finally, macrophages were visualized by fluorescence microscopy (DFC310, LECI, Germany).
For the intracellular ROS detection, cells were pretreated with extract or MgCl2 for 1 h and then stimulated with LPS for 1 h. Next, cells were stained using dichlorodihydrofluorescein diacetate (DCFH, Beyotime, China) according to the protocol. Finally, THP-1 cell-derived macrophages were harvested and analyzed by flow cytometry [fluorescence-activated cell sorting (FACS); Canto II, BD, USA] or directly visualized using a fluorescence microscope.
For the TLR-4 detection, cells were pretreated with extract or MgCl2 for 1 h before LPS stimulation for 24 h. Cells were washed with PBS and stained with TLR-4-PE (BioLegend, USA) for 30 min. After that, the results were analyzed with FACS.
The FACS data were processed using Flowjo 7.6 software.
For the intracellular ROS detection, cells were pretreated with extract or MgCl2 for 1 h and then stimulated with LPS for 1 h. Next, cells were stained using dichlorodihydrofluorescein diacetate (DCFH, Beyotime, China) according to the protocol. Finally, THP-1 cell-derived macrophages were harvested and analyzed by flow cytometry [fluorescence-activated cell sorting (FACS); Canto II, BD, USA] or directly visualized using a fluorescence microscope.
For the TLR-4 detection, cells were pretreated with extract or MgCl2 for 1 h before LPS stimulation for 24 h. Cells were washed with PBS and stained with TLR-4-PE (BioLegend, USA) for 30 min. After that, the results were analyzed with FACS.
The FACS data were processed using Flowjo 7.6 software.
4
Immunophenotyping of Hematopoietic Cells
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A single-cell suspension of approximately 107 cells/ml was prepared, and 100 μl was added to a flow tube. Five µL of each reagent (CD38 FITC, CD138 PE, CD27 APC)/(CXCR4 APC, TLR4 PE) were added to the cell suspension, incubated in the dark for 30 min, and centrifuged at 400 × g for 5 min at room temperature. The supernatant was discarded, 2 mL of PBS was added, and the sample was centrifuged again at 400 × g for 5 min at room temperature, with a subsequent repetition of these processes. After centrifugation, 500 µL of PBS and a flow cytometer probe (NovoCyte 2060R, ACEA BIO Co., Hangzhou, China) were added for detection. CD38 FITC, CD138 PE, CD27 APC, CXCR4 APC, and TLR4 PE were purchased from BioLegend (San Diego, CA, USA).
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