was measured in vitro as the capacity of monocytes to express IL-6, using the 1130h blood sample on each of the seven intensive recording days. Whole blood was stimulated with lipopolysaccharide (LPS) from Escherichia coli 0127-B8 (LPS 100pg/ml, Sigma-Aldrich), and then brefeldin A (10 ug/ml, Sigma-Aldrich) was added to the sample, which was incubated for 4 hours at 37 °C in a 5% CO2 atmosphere. Following fixation and permeabilization procedures (Intraprep™ Permeabilization reagents [Beckman Coulter]), fluorescence-conjugated antibodies were added (CD14 APC, CD45 KrO [both Beckman Coulter], IL-6 PE [BD Bioscience]) and samples incubated for 15 min at room temperature in the dark. Samples were vortexed, washed with phosphate-buffered saline solution (PBS 1X, Sigma Aldrich), and stored at 2–8 °C in the dark after re-suspension in 500 ul of PBS containing 0.5% formaldehyde. Preparations were analyzed within 24 hours using a Gallios™ flow cytometer (Beckmann-Coulter) at the Flow Cytometry Core at BIDMC, and 100,000 events were acquired. Percentage of IL6-positive monocytes (LPS-stimulated and spontaneous) were quantified using Kaluza® Flow Analysis software (Beckmann Coulter).
Il 6 pe
Manufactured by BD
IL-6 PE is a laboratory equipment product that measures the expression of the Interleukin-6 (IL-6) protein using the phycoerythrin (PE) fluorescent label. It is used for quantitative analysis and detection of IL-6 in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Gallios flow cytometer, by Beckman Coulter (2 mentions)
Cd14 apc, by Beckman Coulter (2 mentions)
Cd45 kro, by Beckman Coulter (2 mentions)
Golgiplug, by BD (2 mentions)
Cytofix cytoperm solution, by BD (2 mentions)
Alexa fluor 647 il 1β, by BioLegend (2 mentions)
Brefeldin a, by Merck Group (1 mentions)
Kaluza flow analysis software, by Beckman Coulter (1 mentions)
Pbs 1x, by Merck Group (1 mentions)
Lps from escherichia coli 0127 b8, by Merck Group (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Fix perm kit, by BD (1 mentions)
Compbead, by BD (1 mentions)
Automacs, by Miltenyi Biotec (1 mentions)
Brefeldin a, by BD (1 mentions)
Lrsii, by BD (1 mentions)
B cell isolation kit 2, by Miltenyi Biotec (1 mentions)
Cd23 pe cy7, by BD (1 mentions)
Imiquimod, by InvivoGen (1 mentions)
5 protocols using il 6 pe
1
Monocyte IL-6 Expression Assay
2
Glucocorticoid Sensitivity in Monocytes
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GC sensitivity was determined by the capacity of the synthetic glucocorticoid DEXamethasone (DEX) to suppress IL-6 expression in monocytes using the 1130 baseline blood sample. Whole blood was stimulated with lipopolysaccharide (LPS) from Escherichia coli 0127-B8 (LPS 100 pg/ml, Sigma-Aldrich), and then different concentrations of DEX (0, 12.5, 25, 50, 100, and 200 nM; Sigma- Aldrich) as well as brefeldin A and fluorescence-conjugates antibodies (CD14 APC, CD45 KrO [both Beckman Coulter], IL-6 PE [BD Bioscience]) were added to the samples. Samples were incubated for 4 hours at 37 °C at 5% CO2. The samples were analyzed the following day in a Gallios flow cytometer (Beckman Coulter Fullerton, CA, Flow Cytometry Core at BIDMC) using Kaluza software (for details, see Simpson et al., 2016 (link)).
3
PBMC Isolation and Stimulation Assay
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PBMCs were isolated from whole blood using Ficoll (Lymphocyte® Cell Separation Media, Mediatech) gradient centrifugation. Cryopreserved PBMC from HIV-infected subjects were used in the immunophenotyping experiments. The cells were stained with FcRL4-APC and CD19-PE-Texas Red and CD19+FcRL4pos and CD19+FcRL4neg B cells were FACS purified and cultured overnight in the presence of 10 µg/ml CpG-B ODN2006 (TLR9L), 2 µg/ml PAM3CSK4 (TLR2L), or 2 µg/ml Imiquimod (InvivoGen). B cells (CD19+) from healthy controls were purified from PBMC using the B Cell Isolation Kit II (Miltenyi Biotec) and AUTOmacs (Miltenyi Biotec). After 4H, the cultures of CD19+ B cells were supplemented with Brefeldin A (1:1,000, BD). After overnight incubation, the cells were surface stained (CD23-PE-Cy7, BD Biosciences), fixed/permeabilized (Fix/Perm Kit BD Biosciences), and stained for intracellular IL-6 (IL-6-PE, BD Biosciences). All samples were acquired on an LRSII (BD Biosciences) flow cytometer and the data analyzed using FlowJo software (Tree Star Inc.). Florescence parameters were normalized using Rainbow Calibration Particles (Spherotech) and antibody bound CompBead (BD Biosciences). Gating was determined by unstained controls.
4
Cytokine Profiling in A549 Cells
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A549 cells (3 × 105) were treated with LAD2 culture supernatant (250 μL) for 24 h, then cells were harvested. The cytokines were determined either by quantifying the production of mRNAs or intracellular immuno-staining with specific antibodies. For immuno-staining assay, A549 cells were added leukocyte activation cocktail containing BD GolgiPlug (BD, 550583) and cultured for 6 h. Following activation, cells were washed with FACS buffer. BD Cytofix/Cytoperm solution (BD, 554722) was used for the simultaneous fixation and permeabilization of cells for 20 min at 4 °C before intracellular cytokine staining. Antibodies diluted in Perm/Wash buffer was added and cells were further incubated at 4 °C for overnight. After washing, cells were resuspended in FACS buffer to flow cytometric analysis (BD LSRFortessa). Cytokine antibodies against the flowing markers were used: Alexa Fluor 647-IL-1β (Biolegend, JK1B-1), PE-IL-6 (BD, MQ2-6A3), BV421-IL-8 (BD, G265-8).
5
Cytokine Profiling in A549 Cells
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A549 cells (3×10 5 ) were treated with LAD2 culture supernatant (250 μL)
for 24 h, then cells were harvested. The cytokines were determined either by quantifying the production of mRNAs or intracellular immuno-staining with specific antibodies. For immuno-staining assay, A549 cells were added leukocyte activation cocktail containing BD GolgiPlug (BD, 550583) and cultured for 6 h. Following activation, cells were washed with FACS buffer. BD Cytofix/Cytoperm solution (BD, 554722) was used for the simultaneous fixation and permeabilization of cells for 20 min at 4°C before intracellular cytokine staining. Antibodies diluted in Perm/Wash buffer was added and cells were further incubated at 4°C for overnight. After washing, cells were resuspended in FACS buffer to flow cytometric analysis. Cytokine antibodies against the flowing markers were used: Alexa Fluor 647-IL-1β (Biolegend, JK1B-1), PE-IL-6 (BD, MQ2-6A3), BV421-IL-8 (BD, G265-8).
for 24 h, then cells were harvested. The cytokines were determined either by quantifying the production of mRNAs or intracellular immuno-staining with specific antibodies. For immuno-staining assay, A549 cells were added leukocyte activation cocktail containing BD GolgiPlug (BD, 550583) and cultured for 6 h. Following activation, cells were washed with FACS buffer. BD Cytofix/Cytoperm solution (BD, 554722) was used for the simultaneous fixation and permeabilization of cells for 20 min at 4°C before intracellular cytokine staining. Antibodies diluted in Perm/Wash buffer was added and cells were further incubated at 4°C for overnight. After washing, cells were resuspended in FACS buffer to flow cytometric analysis. Cytokine antibodies against the flowing markers were used: Alexa Fluor 647-IL-1β (Biolegend, JK1B-1), PE-IL-6 (BD, MQ2-6A3), BV421-IL-8 (BD, G265-8).
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