Trypsin 0.03 edta

The semi-adherent JAWSII cell line, a granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent dendritic (DC) line established from bone marrow cells of p53-knockout C57Bl/6 mouse, was purchased from the American Type Culture Collection (CRL-1194; ATCC, Manassas, VA). JAWSII cells were maintained in complete Roswell Park Memorial Institute medium (RPMI) 1640 culture with 4 mM of l-glutamine, HEPES (Thermo Fisher Scientific) consisting of 10% (v/v) fetal bovine serum (FBS), 5 ng/mL of GM-CSF, 10 U/mL of penicillin and 100 μg/mL of streptomycin, 0.5 mM of 2-ME, and 1 mM of sodium pyruvate, in 5% CO₂ in air at 37°C. Cultures were maintained by transferring nonadherent cells to a centrifuge tube and treating attached cells with 0.25% trypsin−0.03% EDTA (Gibco) at 37°C for 5 min, followed by pooling the two populations of cells together and dispensing into new flasks and/or for downstream analysis. To stimulate JAWSII cells, 300,000 cells/mL were seeded into wells of 6-well plates for 24 h before exposure to liver fluke Hz, OfHz. The cells were divided into three groups: control (no treatment), mock (1 × PBS treatment), and OfHz-treated (100 nm of OfHz in 1 × PBS). JAWSII cells were pulsed with OfHz for 48 h and harvested as above. The cells were washed three times with 1 × PBS before proceeding to RNA extraction.

Hematopoietic stem cells from femurs and tibias of wild-type and knockout mice were used to generate bone marrow-derived DCs (bmDCs) as previously described [11 (link), 14 (link)] in RPMI-1640 supplemented with 5% heat-inactivated fetal bovine serum, 10 mM HEPES, 2 mM l-glutamine, and 50 µM 2-mercaptoethanol (all from Gibco, Burlington, ON, Canada) and complemented with 10% granulocyte–macrophage colony-stimulating factor. For macrophages (MΦ), hematopoietic stem cells (5 × 10⁵ cells/mL) were cultured in Dulbecco’s Modified Eagle’s Medium supplemented with 10% heat-inactivated fetal bovine serum (Gibco) and complemented with 30% L929 cell-derived macrophage colony-stimulating factor supernatant [34 ]. Cells were cultured for 8 days at 37 °C with 5% CO₂ and trypsinized using 0.05% trypsin-0.03% EDTA (Gibco) prior to infection. Cell purity was at least 85% CD11c⁺ or F4/80⁺ for bmDCs and MΦ, respectively. Albeit this culture system cannot completely rule out the presence of other innate cells (such as macrophages), it represents an enriched source of bmDCs.

An immortalized immature dendritic cell line, JAWS II (ATCC, Manassas, VA, USA), was used as a model of immune responsive cells. Cells were cultured in a complete culture medium consisting of IMDM with 10% FCS, 4 mM L-glutamine, 10 U/mL penicillin and 100 μg/mL streptomycin, 0.5 mM 2-ME, and 1 mM sodium pyruvate. Nonadherent cells were transferred to a centrifuge tube and the attached cells were treated with 0.25% trypsin 0.03% EDTA (Gibco) at 37 °C for 5 min and transferred to the centrifuge tube. Then the two populations were transferred to a new flask. Before cell inoculation, the cell culture chips were washed thoroughly with 70% ethanol, rinsed with distilled water, exposed to UV light for at least 30 min, and incubated with cell culture media overnight in a cell culture incubator (37 °C with humidified atmosphere and 5% CO₂). JAWS II cells were inoculated into the lower compartment via the corresponding fluidic inlet and cell-free media was injected into the upper compartment. Fresh culture media was perfused into the chip through the syringe pump at a constant flow rate of 20 nL/s. To quantify cell viability, cells were mixed with trypan blue at a 1:1 ratio and counted for living and dead cells using an automated cell counter (Millipore, Burlington, MA, USA).