Mouse tgf beta 1 duoset elisa

Protein concentration of TGFβ in BALF was analyzed by ELISA (Mouse TGF-beta 1 DuoSet ELISA; DY1679; R&D Systems, Minneapolis, MN, USA). The snap frozen supernatants of BALF were thawed and ELISA was carried out according to the manufacturer’s instructions. The undiluted samples were activated using a sample activation kit (DY010; R&D Systems, Minneapolis, MN, USA). Duplicates of standards and samples were transferred to a 96-well plate and the protein concentration was determined spectrophotometrically at 450 nm using a microplate-reader (Spark 10 M, Tecan).

Mice were anaesthetized with Isoflurin and transcardially perfused with PBS. Spleen was dissected, and splenocyte suspension was prepared according to Majer et al. (2020 ). Briefly, the spleen was homogenized, filtered through a 70 μm cell strainer and got rid of red blood cells with an ACK lysis buffer. Splenocytes were counted using the Countess Automated Cell Counter (ThermoFisher Scientific, Waltham, USA). Two million splenocytes per well (2 million cells mL⁻¹) were cultivated in RPMI 1640 supplemented as described in Majer et al. (2020 ) in 12-well plates and stimulated with either MOG (10 or 50 μg mL⁻¹), concanavalin A (1.25 μg mL⁻¹) or left untreated for 72 h. Concentrations of interferon (IFN)-γ, IL-17, IL-1β, IL-4, IL-5 and IL-10 were measured in the culture supernatants using ELISA MAX Standard kits (BioLegend) according to the manufacturer's protocol. The concentration of transforming growth factor (TGF)-β was measured in the culture media using Mouse TGF-beta 1 DuoSet ELISA (R&D Systems, Minneapolis, USA) according to the manufacturer's protocol.

BALF was obtained from mouse lung by flushing the lungs with PBS and spinning down the fluid to remove the cells. A Mouse TGF-beta 1 DuoSet ELISA (R&D Systems, Minneapolis, MN) was used according to manufacturer’s instruction to measure immunoreactive TGβ1.