Gm csf

Chicken BMDCs were generated and expanded in vitro by culturing bone marrow precursors with 50 ng/ml recombinant chicken granulocyte colony-stimulating factor (GM-CSF, Abcam, USA) and 50 ng/ml IL-4(Kingfisher, USA), as described [20 (link), 21 (link)]. At day 6, the non-adherent, relatively immature DCs were harvested and placed in fresh medium (1 × 10⁶ cells/ml) with LPS (1 μg/ml) stimulation. Cells were then collected, washed and incubated at 4 °C for 30 min with PE-conjugated anti-human CD11c (0.05 mg/ml), FITC-labeled anti-chicken major histocompatibility complex class II (MHC-II) antibody (0.5 mg/ml). After three times washing, cells were analyzed with Fluorescence Activated Cell Sorter (FACS) (BD, FACS Aria) (Additional file 1). Cells were incubated with IBDV at a multiplicity of infection of 1 (MOI = 1) for 12 h.

Primary bone marrow (BM) cells were isolated and cultured according to the published procedures [44 , 45 (link)]. Femurs and tibiae of naïve BALB/c OlaHsd (Envigo RMS BV, Horst, the Netherlands) mice were cut with scalpel from each end and flushed with cold PBS to collect BMs, which were passed through 70 μm cell strainers (Becton-Dickinson, BD, Franklin Lakes, NJ, USA). The single cell cultures were suspended in complete medium (CM, RPMI-1640 supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, 50 μm 2-mercaptoethanol, 2 mm l-glutamine, and 10% fetal bovine serum (FBS), all from Sigma-Aldrich) and subjected to centrifugation for 10 min, 300×g. The cells were seeded on nontreated 14.2 mm sterile petri dishes (VWR, Radnor, PA, US) at 1 × 10⁶ cells/ml (10 ml/plate) in CM/10% FBS supplemented with a recombinant mouse granulocyte-macrophage colony-stimulating factor (GM-CSF, Abcam, Cambridge, UK) at 20 ng/ml and cultured for 8 days (37°C and 5% CO₂). Five milliliters of fresh medium containing GM-CSF (20 ng/ml) was added on days 4 and 7, and nonattached cells were harvested from plates with gentle washing on day 8. The cells were tested for the expression of CD11c molecules, costimulation molecules (CD80, CD86), and MHC class II (I-A/I-E) by surface staining and flow cytometry as described below.

The levels of cytokines (IL-1RA, IL-10, CCL2, IFNα, IFNγ, GM-CSF, and TNFα; purchased from Abcam, Cambridge, USA) were measured and compared in media collected from the LOMM models. For mature IL-1β, we used the IL-1 beta Human ELISA Kit (Thermo Fisher Scientific, USA, Cat# BMS224-2). We also measure the cytokine level in media collected from the 3D-BSGM models per the manufacturers' instructions.

Bone marrow cells were obtained from the femurs and tibias of NOD/Ltj mice, and red blood cells were eliminated using ACK Lysis Buffer (Beyotime, China). Then bone marrow cells were washed and cultured in six-well tissue culture plates at 1×10⁶ cells/mL in complete RPMI culture medium supplemented with 10% fetal bovine serum and GM-CSF (20 ng/ml, Abcam) at 37°C, 5% CO2. The culture medium was changed on culture day 2 and 4. Fresh medium and GM-CSF were added after flushing out non-adherent cells. On day 6, loosely adherent clustered cells were harvested as immature BMDCs. To detect the ability of sEVs to promote DC maturation, immature BMDCs, cultured in complete medium supplemented with GM-CSF (10ng/ml) and IL-4 (5ng/ml), were treated with HG-sEV and C-sEV (9×10⁹ particles/ml) respectively for 24 h. LPS (1μg/ml) was used as a positive stimulant to promote DC maturation.

We took 2 mL of the rat’s blood at room temperature, centrifuged at 3000 r/min for 15 min at static pressure for 2 h, and then stored at − 80 °C after collecting the supernatant liquid. We performed the procedure in strict accordance with the ELISA Kit manual. We used the double antibody sandwich-ELISA assay test, to determine the IL-5, IL-10, GM-CSF, CCL5, TGF-β, and IFN-γ (Abcam, Cambridge, UK) in the serum.