Human inflammatory cytokines multi analyte elisarray kit

Bone marrow extracellular fluid was obtained by flushing femur and tibia of tumor-bearing mice with 500 μL ice-cold phosphate-buffered saline containing protease inhibitor cocktail (P8340, Sigma-Aldrich, St. Louis, MO), and the supernatant was harvested by centrifugation at 400 g for 5 min. Cytokine levels in the marrow fluids were analyzed by antibody sandwich enzyme-linked immunosorbent assay (Human Inflammatory Cytokines Multi-Analyte ELISArray Kit, MEH004A, QIAGEN, Valencia, CA), according to the manufacturer's protocol. The marrow fluids obtained from non-tumor bearing mice were used as controls. Cytokine levels were normalized to total protein.

QFM measurements of TGF-β1 were validated by comparing results with ELISA and qPCR measurements of muscle homogenates from PAD-II and CTRL patients (N = 13 in each group). TGF-β1 protein expression was measured as part of a customized Human Inflammatory Cytokines Multi-Analyte ELISArray Kit (Qiagen, Valencia, CA, USA).
To measure TGF-β1 gene transcripts in skeletal muscle biopsies, RNA extraction, reverse transcription reactions, and qPCR were performed as previously described [40 (link), 42 (link)] and levels were normalized to myosin gene transcripts. Intrasession reliability was determined by comparing the TGF-β1 measurement of each slide from the mean of its duplicate pair. Intersession reliability was determined from the averages of patient biopsy specimens, analyzed a second time in the next analytical session. Detailed methodologies and validation results can be found in “Additional file 1”.

ELISA experiments were performed using Human Common Chemokines Multi‐Analyte ELISArray Kit (Qiagen MEH‐009A) and Human Inflammatory Cytokines Multi‐Analyte ELISArray™ Kit (Qiagen MEH‐004A), respectively, according to manufacturer protocol. Conditioned media were collected from EpCAM−/CD133− control and 100 nm Dox‐treated EpCAM−/CD133− cells on day 6. Briefly, after centrifugation at 1200 rpm for 5 min, 50 µl of conditioned media was added to each well of the array. Results were normalized with negative and positive controls supplied by the kit. Well absorbance was measured at 450 nm as well as at 570 nm to normalize for optical imperfections using a microplate reader (Thermo Scientific Multiskan Go, USA).

Cytokines and chemokines were measured using the Human Inflammatory Cytokines Multi-Analyte ELISArray kit (Qiagen) and the Human Common Chemokines Multi-Analyte ELISArray Kit (Qiagen) according to the manufacturer's instructions. Briefly, 50 μL of medium was incubated in a 96-well plate for 1 h at room temperature. This was followed by several washing and incubation steps as follows: incubation with detection antibodies for 1 h; wash; incubation with avidin-horse radish peroxidase for 30 min; wash; color development for 15–30 min; development termination using stop solution; and colorimetric quantitation of cytokines or chemokines by reading the optical density at 450 nm. In addition, the concentration of DEFB131 was measured using a DEFB131 ELISA Kit (Uscn Life Science Inc., Wuhan, China) according to the manufacturer’s instructions. Serial dilutions of recombinant peptide provided by the kit were used for generating standard curves. The optical density of the wells was determined using a microplate reader (TECAN System Inc., Männedorf, Switzerland) set to 450 nm.

The enzyme-linked immunosorbent assay (ELISA) was used to detect IL-8 in the tissue extracts as well as in the culture medium (Human IL-8 CytoSet, Thermo Fisher Scientific Inc., Rockford, IL, USA). The assay was run according to the manufacturer’s instructions. For the determination of other inflammatory cytokines (IFN-γ, TNF-α, GM-CFS, IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12 and IL-17A), the protein extracts from three donors were pooled, diluted 2-fold and analyzed using a Human Inflammatory Cytokines Multi-Analyte ELISArray Kit (Qiagen, Hilden, Germany).

Supernatants were obtained from MSC culture as well as MSC–T-cell co-cultures.
IL-6, TNF-α, and IL-4 levels were measured using a commercially available Human Inflammatory Cytokines Multi-Analyte ELISArray Kit (Qiagen, QIAGEN S.p.A., Italy) according to the manufacturer’s instructions. Briefly, a microplate was coated with monoclonal antibodies that were specific to the cytokines. Standards and supernatants were pipetted into the wells of the microplate. A positive control was obtained by pipetting only the standard into the wells. A negative control was obtained by pipetting the standard and cell culture supernatants into non-coated wells. After the plate was washed, enzyme-linked polyclonal antibodies specific for IL-6, TNF-α, and IL-4 were added to the wells. The reaction was revealed by the addition of the substrate solution. The optical density was measured at a wavelength of 450 nm by using the Tecan Infinite M200 (Tecan, Switzerland) spectrophotometer. Cytokines concentrations (pg/mL) were determined against a standard concentration curve. All samples were run in duplicate.