Pore size transwell inserts

To determine whether addition of macrophages to the culture system enhanced parasite survival further, a monocyte-derived cell line, THP-1 (ECACC), originally derived from an acute monocytic leukaemia patient were evaluated. To differentiate THP1 cells into macrophages (Mϕ), cells were treated with 10 ng/ml of 12-O-tetradecanoylphorbol-I3-acetate (PMA; Peprotech), as previously described [22 (link)]. For polarisation into either a classically- or alternatively-activated phenotype, cells were treated with 50ng/ml recombinant interferon-gamma, (IFN-γ), or 25 ng/ml of recombinant interleukin 4 (IL-4) and interleukin 13 (IL-13), respectively (Peprotech), as previously detailed [23 (link)]. Forty-eight hours post-stimulation, Mϕ(IFN-γ), Mϕ(IL-4/IL-13), or non-polarised Mϕ(naïve) cells were washed 3 times in fresh medium and 0.4 μm pore-size transwell inserts (Corning) were added to fresh monolayers of LECs in a 6-well plate. Six millilitres of EGM-2 MV medium was then added, ready for the addition of parasites, as detailed above. Lymphatic endothelial cell monolayers with additional LEC trans-well inserts were included as controls.

Transepithelial resistance measures the integrity of cellular tight junctions and is a suitable method to be used in cell culture monolayers for the purpose of measuring the effect of bacteria during infection in vitro (Srinivasan et al., 2015 (link)). This methodology measures the electrical resistance, in ohms, as a measure of cellular barrier integrity. For the purpose of TEER measurement, the HCT-8 cells were grown on 0.4 μm and 12 mm pore size transwell inserts (Corning) and selected based on the formation of a confluent monolayer. Our aim was to investigate the effect of Auranta 3001 on the barrier properties of HCT-8 cells by taking TEER measurements at 3 h postinfection (±0.1 and 0.5% Auranta 3001) using an EVOM X meter connected to an Endohm chamber (World Precision Instruments).

The migration of M-07e was conducted in a transwell system. The lower surfaces of the filter in 8.0 μm pore size transwell inserts with 6.5 mm diameter (Corning, USA). We seeded 1 × 10⁴ M-07e in 200 μl serum-free IMDM with 10 ng/ml GM-CSF onto the upper transwell compartments in 24-well plates. The normal or BLM-induced lung tissue on day 7 after BLM administration was harvested, cut into pieces (1 mm³), and incubated into the lower chamber, with/without WZ811 (1 μmol/L, Selleck, USA) added to the upper chambers to treat M-07e. The cells were allowed to migrate for 12 h. After washing and removing the remaining M-07e on the upper surface of the filter with a swab, the migrated M-07e were observed and counted after 0.1% crystal violet staining (Solarbio, China) and photographed with a microscope (Nikon, Japan).

The coculture systems were established through 6-well plates and 0.4 μm pore size transwell inserts (Corning) [24 (link)]. Anti-Act1 or wildtype BMDMs (1 × 10⁶ cells/well) were seeded in 6-well plates and two CRC cell lines, CT26 or MC38 cells (5 × 10⁵/insert), were seeded in transwell inserts. Another coculture system exchanged the position of CT26 or MC38 cells with macrophages. For convenience, the aforementioned cells located in lower plates were regarded as research objects in the following experiments. For CD8⁺ T cells isolation, coculture with BMDMs and CRC cell lines, and flow cytometry analysis, the detailed methods were present in Additional file 3: Supplementary methods.