The secretion of Interleukin 6, Interleukin-2 and IFN-γ in co-cultures of cells with hPBMCs were evaluated by ELISA assays. Briefly, after treatments culture supernatants were centrifuged and treated for quantification of human IL-6 (ELISA MAXTM Deluxe Set Human IL-6, BioLegend, San Diego, CA, USA), of IL-2 and IFN-γ (DuoSet ELISA, R&D Systems, Minneapolis, MN, USA), according to the producer’s recommendations. Concentration values were reported as the mean of at least three determinations.
Elisa maxtm deluxe set human il 6
Manufactured by BioLegend
Sourced in United States
The ELISA MAXTM Deluxe Set Human IL-6 is a laboratory equipment product designed for the quantitative measurement of human interleukin-6 (IL-6) in biological samples.
Automatically generated - may contain errors
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Duoset elisa, by R&D Systems (2 mentions)
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Elisa maxtm deluxe set human mcp 1 ccl2, by BioLegend (1 mentions)
Infinite 200 pro, by Tecan (1 mentions)
Mycoplasma detection kit, by Lonza (1 mentions)
Erk1 2, by Cell Signaling Technology (1 mentions)
5 protocols using elisa maxtm deluxe set human il 6
1
Cytokine Secretion in Co-Cultures
2
Cytokine Secretion Evaluation in Cell Cocultures
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Supernatants of co-cultures of cells with hPBMCs were analysed by ELISA assays to evaluate the secretion of interleukin 6, interleukin-2, IFN-γ, and granzyme B. Briefly, after the treatments in the absence or presence of immunomodulatory mAbs, supernatants were centrifuged and treated for quantification of human IL-6 (ELISA MAXTM Deluxe Set Human IL-6, BioLegend, San Diego, CA, USA), human IL-2, IFN-γ, and granzyme B (DuoSet ELISA, R&D Systems, Minneapolis, MN, USA), according to the producer’s recommendations. ELISA assays to measure TNF-α cytokine level were performed by using Ella Automated Immunoassay system (R&D Systems, Minneapolis, MN, USA), following the producer’s recommendations. Concentration values were reported as the mean of at least three determinations.
3
Anti-inflammatory Activity of Lipid-based Nanoparticles
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In order to evaluate the anti-inflammatory activity of the CU- and RV-based SLNs, containing or not containing LNA, NCTC 2544 cells were seeded at the concentration of 250 × 103 cells/well in a 12-well multiwell culture plate. After 24 h, cell culture medium was removed and replaced with fresh culture medium containing or not the empty SLNs (containing only CU or RV) and SLNs loaded with LNA (at a final concentration of 5 µg/mL) in the absence and in the presence of TNF-α (20 ng/mL), as a pro-inflammatory stimulus. At the end of the treatment period (24 h), surnatant was collected and stored at −80°C until the evaluation of the cytokine secretion. The presence of the pro-inflammatory cytokines IL-6 and MCP-1 were evaluated by ELISA assay using commercially available kits (ELISA MAXTM Deluxe Set Human IL-6, cat. 430504; ELISA MAXTM Deluxe Set Human MCP-1/CCL-2, cat. 428804, Bio-Legend, San Diego, CA, USA) following the instructions of the manufacturer. The minimum level of measurable cytokines was 4 pg/mL and 3.9 pg/mL, for IL-6 and MCP-1, respectively.
4
Quantifying IL-6 Secretion in Cancer Cells
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To analyze IL-6 secretion, cancer cells (8 × 105) were seeded in a 60-mm culture dish with complete medium and incubated for 72 h. The complete medium was removed and replaced with 2 ml serum-free medium, and the cells were incubated for 48 h. The medium was collected by centrifugation at 2,000 rpm and 4 °C for 10 min. The supernatants were immediately used for analysis of IL-6 secretion by the Human IL-6 ELISA MAXTM Deluxe Set (#430505, BioLegend, San Diego, CA, USA) according to the manufacturer’s protocol. Reactions were stopped with 2 N H2SO4, and the absorbance was read at 450 nm and 570 nm using a Tecan Infinite 200 PRO multimode microplate reader (Tecan). The absorbance of samples at 570 nm was subtracted from the absorbance at 450 nm.
5
Profiling HMCL Models for Drug Screening
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All HMCLs used in this study were provided by Dr. Leif Bergsagel’s laboratory. All cell lines were fingerprinted using CNV analysis to confirm their identity as described20 . Cells were maintained in RPMI-1640 media, supplemented with 5% fetal calf serum and antibiotics. All HMCLs tested negative for mycoplasma at the beginning and during the experiments (using mycoplasma detection kit from Lonza, Rockland, ME). XG1 was originally reported as an IL-6-dependent cell line21 (link), however, the strain that we worked with also grew well in its absence. Antibodies against IRF4 (#4964), PARP (#9542), BIM (#2819), p-STAT3 (#9145), STAT3 (#9139), p-ERK1/2 (#9101), and ERK1/2 (#4695) were from Cell Signaling Technology (Danvers, MA). Anti-MYC (#ab32072) antibody was from Epitomics (Burlingame, CA). Anti-CRBN (#HPA045910) antibody was from Sigma-Aldrich (St. Louis, MO). Anti-IKZF3 (#img-6283a) was from Imgenex (Littleton, CO). Anti-IKZF1 (#sc-13039), Anti-CD147 (8D6) (#sc-21746), and MCT1 (H70) (#sc-50324) antibodies were from Santa Cruz Biotechnology (Dallas, TX). Lenalidomide (Len) and bortezomib (Bor) were from LC Laboratories (Woburn, MA). PB-1-102 (PB) was from Selleckchem (Houston, TX). CC-220 and SGC-CPB30 (SGC) was from MedChem Express (Monmouth junction, NJ). Human IL6 ELISA MAXTM Deluxe set was from Biolegend (San Diego, CA).
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