Human gm csf quantikine elisa kit

IL-18 and IL-12 levels in the serum and GCF were measured by Human Total IL-18 Quantikine QuicKit ELISA (R&D Systems) and Human IL-12 p70 Quantikine ELISA kit (R&D Systems), respectively. Interferon-γ (IFN-γ), granulocyte macrophage-colony stimulating factor (GM-CSF), IL-2, and tumor necrosis factor-α (TNF-α) levels in the cultured supernatants were measured by Human IFN-γ Quantikine ELISA kit (R&D Systems), Human GM-CSF Quantikine ELISA kit (R&D Systems), Human IL-2 Quantikine ELISA kit (R&D Systems), and Human TNF-α Quantikine ELISA kit (R&D Systems), respectively.

Milk was collected with a pipette from narcotized female mice at day 10 of lactation after oxytocin administration and stored at − 80 °C. For ELISA analysis, milk was diluted 1–6 million times with water and assayed according to the manufacturer’s recommendation (Human GM-CSF Quantikine ELISA Kit, DGM00, R&D Systems). Blood was collected from the euthanized mice and the serum was diluted × 1000 times for the assay. Measurements were taken at 490 nm with BioTek Epoch Spectrophotometer.
The protein concentrations in milk were quantified using Pierce BCA Protein Assay Kit (ThermoFisher). For Western blot, equal amounts (20 μg) of milk samples were separated on 15% SDS-PAGE, and then transferred onto Immun-Blot PVDF membrane (Bio-Rad). Membrane was blocked with 5% milk/TBST(20 mM Tris pH 7.5, 150 mM NaCl, 0.1% Tween 20) for 2 h and incubated with primary antibodies against hGMCSF 1:500 at 4 °C overnight (R&D, catalogue # AF-215-NA). Next day, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies 1:1000 (#93702, Cell Signaling) for 2 h at 25 °C. Detection was performed with SuperSignal West Pico PLUS Chemiluminescent Substrate (#34580, ThermoFisher) and Chemidoc XRS Imaging system (Bio-Rad). Reversible staining of the PVDF membrane (Fig. 3A) was performed with Pierce Reversible Protein Stain Kit (#24585, ThermoFisher).

The concentration of G-CSF or GM-CSF in all samples was evaluated using an ELISA kit (R&D Systems, USA, Human G-CSF Quantikine ELISA Kit and Human GM-CSF Quantikine ELISA Kit), following the manufacturer’s instructions. To ensure measurements within the detection range of the ELISA kits, GM-CSF samples were diluted at a 1:5 ratio using the dilution buffer provided with the ELISA kit. Each sample was analyzed in duplicate during the ELISA analysis.

ELISA was performed to quantify the level of GM-CSF present in MCF7 and MDA-MB-231 cells with altered FRG1 expression. In total, 1 × 10⁶ cells were plated in a 100 mm culture dish in their respective culture media. After 12 h, the media was replaced by the media containing 2% FBS. After 3 days of incubation, the media was collected and centrifuged at 4000 rpm (4 °C) for 10 min to get rid of the cellular debris. The supernatant was aliquoted and stored at −80 °C till further use. 100 μl of this supernatant was used to carry out the ELISA using Human GM-CSF Quantikine ELISA Kit (R&D Systems, MN, USA) according to the manufacturer’s protocol. OD values were taken at 450 nm and 540 nm in the Varioscan multimode microplate reader (Thermo). During the final analysis, values obtained at 450 nm was subtracted from the values at 540 nm.