TLR4 endocytosis was determined as described [25 (link)]. Briefly, primary mouse hepatocytes were treated with SAA1 (2 μg ml−1) or control for 120 min at 37 °C. Cells were subsequently stained with the antibody against TLR4 (TLR4-APC, SA15-21, BioLegend) for 30 min on ice. The cells marked by the antibody were washed three times with PBS and analyzed with FACS Verse (BD Biosciences). The geometric mean fluorescence intensity of TLR4-APC was recorded.
Sa15 21
Manufactured by BioLegend
SA15-21 is a compact and lightweight tabletop centrifuge with a maximum speed of 15,000 rpm. It is designed for a variety of general laboratory applications that require centrifugation.
Automatically generated - may contain errors
Lab products found in correlation
Tlr4 apc, by BioLegend (1 mentions)
Facsverse, by BD (1 mentions)
Anti cd19 apc 6d5, by BioLegend (1 mentions)
Anti cd3 fitc 17a2, by Thermo Fisher Scientific (1 mentions)
Anti cd11c pe n418, by Thermo Fisher Scientific (1 mentions)
Anti cd11c percp cyanine5, by Thermo Fisher Scientific (1 mentions)
Saponin, by Merck Group (1 mentions)
Facscanto flow cytometer, by BD (1 mentions)
3 protocols using sa15 21
1
TLR4 Endocytosis in Mouse Hepatocytes
2
TLR4 Internalization Kinetics
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Cells were stimulated with 100 ng/ml LPS at 37°C for the indicated times. TLR4 internalization was terminated by adding ice-cold 0.1% azide–containing medium and stored on ice. Surface-remaining TLR4 was detected by staining with an anti–mouse TLR4 antibody (SA15-21; BioLegend). TLR4 binding by clone SA15-21 is not interrupted by LPS stimulation. Cells were then fixed with 2% paraformaldehyde before analysis by flow cytometry.
3
Multiparametric Flow Cytometry of NALT Cells
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Single-cell suspensions of NALT cells were stained for 30 min at 4°C with appropriate combinations of fluorochrome-conjugated maternal antibodies in the presence of 0.2% bovine serum albumin (BSA) and fixed in 4% paraformaldehyde in PBS. Cells were stained for surface markers with anti-CD284 (TLR4)-PE-Cyanine7 (SA15-21, Biolegend) or anti-CD284 (TLR4)-PE (SA15-21, Biolegend), anti-CD11c-PE (N418, eBioscience), or anti-CD11c-PerCP-Cyanine5.5 (N418, eBioscience), anti-CD19-APC (6D5, Biolegend), anti-CD3-FITC (17A2, eBioscience), anti-CD11b-PE-Cyanine7 (M1/70, eBioscience), anti-Ly-6G/Ly-6C (Gr-1)-APC (RB6-8C5, Biolegend), anti-F4/80-PE (BM8, eBioscience), and anti-CD282 (TLR2)-PE (CB225, Biolegend) as needed. For intracellular staining, fixed cells were permeabilized and stained in saponin (0.1% in PBS; Sigma) with anti-CD289 (TLR9)-FITC (M9.D6, eBioscience) and anti-CD283 (TLR3)-PE (11F8, Biolegend) in the presence of 1.5% BSA. Samples were analyzed using a FACSCanto flow cytometer (BD Biosciences) and FlowJo software (Treestar, Ashland, OR).
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