Human gm csf duoset elisa

Breast cancer cell CM samples were collected and used to perform a human GM-CSF DuoSet ELISA (R&D Systems). Murine tumor cell CM was used to perform mouse GM-CSF ELISA (R&D Systems). STAT5^fl/fl DCM and STAT5^cKO DCM was subjected to mouse Type I Collagen ELISA (Novus Biologicals). All ELISAs were performed according to the manufacturer’s instructions.

Supernatants from each experiment were harvested a day before adding thymidine and frozen at −20 °C. Cytokines were measured using ELISA for human IFNγ ELISA MAX (BioLegend), human GM-CSF DuoSet ELISA (R&D Systems) and bead-based assays. A Synergy H1 hybrid multi-mode reader (Agilent) was used to measure the absorbance. The LEGENDplex Multi-analyte Flow Assay kit (BioLegend) is a 13-plex human T_H cytokine panel, which includes IFNγ, TNF, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-17A, IL-17F, IL-21 and IL-22. Staining was conducted as suggested by the manufacturer. LSR Fortessa Flow Cytometer (BD Biosciences) was used to measure cytokines, and data were analysed using FlowJo (Tree Star). The Human XL Cytokine Luminex Performance Assay Fixed Panel (R&D Systems) contains 45 analytes covering a broad range of various chemokines and cytokines. Bio-Plex 200 systems (Bio-Rad) was used to measure cytokine and chemokine secretion as suggested by the manufacturer.