To measure albumin secretion level of HLO, 200 μL of culture supernatant was collected from HLO embedded in Matrigel. For IL-6 and P3NP, 20-30 organoids were seeded and cultured on an ultra-low attachment multiwell plates 96 well plate (Corning). The supernatants were collected after 24 (for albumin), 96 (for IL-6) or 120 hours (for P3NP) from the culture and stored at −80 °C until use. To define the exact number of organoids in each well, the organoids were captured on The KEYENCE BZ-X710 Fluorescence Microscope. The supernatant was assayed with Human Albumin ELISA Quantitation Set (Bethyl Laboratories, Inc., TX, USA), Human IL-6 ELISA Kit (Biolegend, CA, USA), and Human N-terminal procollagen III propeptide, PIIINP ELISA Kit (My BioSource, CA, USA) according to the manufacturer’s instructions. Secreted level of IL-6 and P3NP was normalize the by the number of organoids in the analyzed well.
Human il 6 elisa kit
Manufactured by BioLegend
Sourced in United States
The Human IL-6 ELISA kit is a quantitative sandwich enzyme-linked immunosorbent assay (ELISA) designed for the measurement of human interleukin-6 (IL-6) in biological samples. The kit utilizes a specific antibody pre-coated microplate and a detection antibody to capture and quantify IL-6 present in the samples.
Automatically generated - may contain errors
Lab products found in correlation
Human albumin elisa quantitation set, by Fortis Life Sciences (1 mentions)
Bz x710 fluorescence microscope, by Keyence (1 mentions)
96 well plate, by Corning (1 mentions)
Human il 8 elisa kit, by Proteintech (1 mentions)
Mouse il 6 elisa kit, by BioLegend (1 mentions)
Hek blue il 1β cells, by InvivoGen (1 mentions)
Mrc 5 cells, by American Type Culture Collection (1 mentions)
Quanti blue, by InvivoGen (1 mentions)
Zeocin, by InvivoGen (1 mentions)
Human tnfα elisa kit, by BioLegend (1 mentions)
8 protocols using human il 6 elisa kit
1
Measuring Organoid Secretion Levels
2
CCC-021-TPP Modulates Cytokine Secretion
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A549 and PC‐14 cells were seeded at a concentration of 250 or 500 cells per well, respectively, in a 96‐well plate in 0.1 mL of DMEM/10% FBS. The next day, DMSO or CCC‐021‐TPP was added. After incubation for 4 days, the medium was changed to 0.1 mL of fresh DMEM/10%FBS, and conditioned media were harvested after 24 hours of cultivation. Cells were lysed in RIPA buffer, and the protein concentration was measured by the BCA method using BSA as a standard. Human IL‐6 and IL‐8 concentrations were measured with the Human IL‐6 ELISA Kit (BioLegend) and the Human IL‐8 ELISA Kit (Proteintech), respectively, according to the manufacturers’ instructions and expressed as ng/mg protein.
3
Multimodal Antibody Profiling of Stem Cells
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The following antibodies were used in this study: APC anti-human CD44v3 antibody (AB5088A, 1:300) was purchased from R&D Systems, (Minneapolis, MN). AF700 anti-mouse/human CD44 antibody (103025, 1:300), FITC anti-human CD81 antibody (cat. 349503, 1:300), APC anti-mouse/human CD44 Antibody (103012, 1:300), PE-Cy7 anti-human CD133 Antibody (393910, 1:300), and APC anti-mouse CD81 (104909, 1:300), and Zombie Aqua (423101), were purchased from Biolegend (San Diego, CA). Human FITC-Heparan Sulfate antibody (H1890-10, 1:300) was purchased from US Biological (Salem, MA). Human IL-6 ELISA kit and Mouse IL-6 ELISA kit were purchased from Biolegend (San Diego, CA).
Neutralizing anti-human IL-6 antibody (mabg-hil6-3) was purchased from In vivogen (San Diego, CA). Heparinase I/III blend (H3917-50UN), chondroitinase ABC (C2905-2UN), and JSI-124 (C4493-1MG) were purchased from Sigma-Aldrich (Saint Louis, MO). PDZ1i was generously provided by Dr. Paul B. Fisher (23 (link)).
Neutralizing anti-human IL-6 antibody (mabg-hil6-3) was purchased from In vivogen (San Diego, CA). Heparinase I/III blend (H3917-50UN), chondroitinase ABC (C2905-2UN), and JSI-124 (C4493-1MG) were purchased from Sigma-Aldrich (Saint Louis, MO). PDZ1i was generously provided by Dr. Paul B. Fisher (23 (link)).
4
Measuring IL-6 Levels in Transfected A549 Cells
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After treated A549 cells by transient transfection for 24 or 48 h, the culture supernatants were collected and the number of cells was counted. The levels of interleukin-6 (IL-6) were analyzed by human IL-6 ELISA kit (Biolegend, San Diego,USA.) according to the manufacturer's protocol and the optical density (OD) was measured at 450 nm with a microplate reader. Finally, IL-6 level was adjusted by the total number of cells.
5
In Vitro IL-1β Neutralization Assay
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MRC-5 cells (ATCC), human lung fibroblasts, were maintained in MEM supplemented with 10% fetal bovine serum (FBS), 1% sodium pyruvate and 2 mM L-glutamine and used for in vitro neutralization assays as described by Economides et al.13 (link) Briefly, cells were seeded in 96-well plates at 3,000 cells/100 µL/well and incubated overnight at 37 °C in a 5% CO2 humidified incubator. Cells were then treated with 4 pM human IL-1β or 60 pM mouse IL-1β in the presence or absence of various concentrations of test antibodies. Eighteen hours after treatment, IL-1β-induced human IL-6 secretion in the supernatant was measured using the human IL-6 ELISA kit (Biolegend) according to the manufacturer’s protocol. HEK-Blue™ IL-1β cells (InvivoGen) were maintained in DMEM supplemented with 10% FBS, 100 µg/mL Zeocin™ and 200 µg/mL hygromycin B. When used for in vitro neutralization assays, cells were seeded in 96-well plates at 5x104 cells/100 µL/well and incubated overnight at 37 °C in a 5% CO2 humidified incubator. Cells were then treated with 4 pM human IL-1β or 40 pM mouse IL-1β in the presence or absence of various concentrations of test antibodies. Eighteen hours after treatment, IL-1β-induced release of secreted embryonic alkaline phosphatase (SEAP) in the supernatant was assessed using QUANTI-Blue™ (InvivoGen) according to the manufacturer’s protocol.
6
Quantifying Inflammatory and Apoptosis Markers
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TNF-α, IL-6, M30, and adiponectin were measured. After blood sampling, centrifugation (3,000 rpm for 10 min; X-22R, Beckman Coulter, CA, USA) was immediately performed and serum samples isolated. The CK18 M30-Apoptosense enzyme-linked immunosorbent assay (ELISA) kit (PEVIVA, Bromma, Sweden) was used to quantify M30 levels in the serum. The adiponectin level was quantified using Human Adiponectin (Acrp30) ELISA Kit (BioLegend, Inc., CA, USA), TNF-α level using Human TNF-α ELISA Kit (BioLegend, Inc., CA, USA), and IL-6 levels using Human IL-6 ELISA Kit (BioLegend, Inc., CA, USA).
7
IL-6 Secretion Measurement in MM Cells
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IL6 in the cell cure media was measured by ELISA assay (Human IL-6 ELISA kit was from Biolegend). Briefly, MM cells were cultured with or without lenalidomide treatment for 48 h. The cell culture media was harvested, diluted, and measured according to the manufacture’s instruction.
8
Serum IL-6 Levels in Trauma Patients
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On arrival patients were assessed based on the Advanced Trauma Life Support (ATLS) principles and scored using the KTS II. Consenting patients had variables age, sex, mode of arrival, duration of injury (time in hours from injury to time blood sample was drawn for analysis of serum IL-6 levels), type of injury, cause of injury and body region injured recorded.
Under aseptic technique, 4mls of venous blood was drawn from a convenient peripheral vein, centrifuged at 1000 r.p.m and the serum layer was removed and stored at -80 °C at the Makerere University College of Health Sciences Immunology Laboratory. Serum assay for IL-6 levels were run at the end of the study period using Human IL-6 ELISA kit supplied by Biolegend [22 ]. Repeated freeze-thaw cycles were avoided as recommended by the manufacturer.
Under aseptic technique, 4mls of venous blood was drawn from a convenient peripheral vein, centrifuged at 1000 r.p.m and the serum layer was removed and stored at -80 °C at the Makerere University College of Health Sciences Immunology Laboratory. Serum assay for IL-6 levels were run at the end of the study period using Human IL-6 ELISA kit supplied by Biolegend [22 ]. Repeated freeze-thaw cycles were avoided as recommended by the manufacturer.
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