Transwell co culture system

Cell migration was assayed using the transwell co-culture system (Corning, Inc., Corning, NY). DBTRG cells were placed on plastic surface of the 24-well plates (1 × 10⁵ cells/well). On the following day, the native MSCs and MSC_TRAIL or MSC_PTEN in serum-free medium were seeded on the microporous membrane (8 μm) of the transwell insert respectively. As described by Nakamizo et al.[51 (link)], MSCs were incubated for 48 hrs at 37°C and 5% CO₂. The inserts were washed with PBS and the upper surface of the membrane was scraped gently using a cotton swab to remove non-migrated cells. Then, the membranes were fixed with 95% ethanol and stained using 0.1% crystal violet. The average number of migrated cells was assessed by counting five randomly selected microscopic fields at 100x magnification. All experiments were done in triplicate. To detect MSCs' migration status with normal cells, DBTRG glioma cells were replaced with native MSCs on the plastic surface of the 24-well plates.

The transwell co-culture system (8 μm pore size, Corning Co., NY, USA) is an advanced method employing semi-permeable membrane inserts that are strategically positioned within cell culture wells, bathed in their corresponding media. For each chamber, a concentration of 200 µl containing 105 cells/ml was meticulously maintained. A total of 96 h was used to co-culture the cells. To evaluate the interaction between podocytes and macrophages, a microfluidic chamber system was employed. Quantification of the cell population was adeptly carried out using the CCK-8 assay, sourced from ThermoFisher Scientific, Beijing, China. Desmosterol was used at a final concentration of 10µM.

EMT was examined with the two-chamber assay using a transwell co-culture system (Corning). RAW 264.7 cells (1 × 10⁴ cells) were seeded into the upper chamber of a transwell culture plate (insert) and MC-38 cells (5 × 10⁴ cells) were seeded on bottom of 24 well plate and incubated with DMEM medium containing 10% FBS for 24hrs. M2-TAM cells were incubated with sf DMEM and treated with 2.5 µM CER or 2.5 µM PA for 48 h. After incubation and treatment with 2.5 µM Cer or PA for 48 h, MC38 cells (1.5 × 10⁴ cells/well in a 12-well plate) were collected, washed with PBS, and incubated with 0.5% BSA in PBS for 45 min. To evaluate whether EMT was downregulated in cells by CER or PA, MC38 cells were labeled with E-cadherin-monoclonal antibody (DECMA-1), PerCP (1:1000) (eBioscience), or Vimentin monoclonal antibody (V9) FiTC (1:1000) (eBioscience). Following a final washing step, the cells were analyzed by flow cytometry.

For inhibition of exosome generation, MSCs were treated with a culture medium containing 10 μM of GW4869 for 24 h (Sigma, USA) and marked as MSC-GW. Next, HCC cell co-culturing with MSCs, MSC-GW cells, or MSC exosomes was conducted with a transwell co-culture system (Corning, USA) based on a previous study (29 (link)). In brief, the HCC cells were seeded into the lower chamber. Then the MSCs or MSC-GW cells were seeded into the upper chamber and co-cultured with HCC cells. MSC exosomes were added into the upper chamber with the isovolumetric medium. The cultured Huh7 and PLC cells were divided into four groups: normal group (without any treatment), MSC group (co-cultured with MSCs), MSC-GW group (co-cultured with MSC-GW), and MSC-Exo group (co-cultured with 20 μg/ml of MSC exosomes). PKH-67 (Sigma, USA) staining was carried out to observe exosome uptake of Huh7 and PLC according to the manufacturer’s instructions. Huh7 and PLC cells were collected for proliferation, apoptosis, invasion, sphere formation, and RT-qPCR assays. Huh7 cells were harvested for RNA sequencing (RNA-seq).

Cell migration was examined with the two-chamber assay using a transwell co-culture system (Corning). CT-26 cells(1 × 10⁴ cells)were seeded into the upper chamber of a transwell culture plate (insert) and RAW 264.7 cells (5 × 10⁴ cells) were seeded on bottom of twenty-four-well plate and incubated with DMEM medium containing 10% FBS for 24 h. Afterwards, CT-26 cells was incubated with sf DMEM and treated with 10 µM CER or 10 µM PA for 48 h. For nuclear staining, the cell suspension in the upper chamber was aspirated and the upper surface of the filter was carefully cleaned with cotton plugs, to remove all cells that did not migrate through the membrane. The nucleus of the cells that migrated through the membrane was stained with DAPI (Thermo Fisher Scientific). Three representative images of two technical replicates were taken with a Leica DM5500 B fluorescence microscope (DAPI filter), equipped with a Leica DFC365 FX digital camera. Digital images were acquired and stored using Leica Application Suite X (LAS X) software. The migratory and invasive cells, which migrated through the 5-μm-sized pores, were counted using Fiji analysis software (https://fiji.sc/).

As described previously,23 1 × 10⁵ cells/well in 200 μL of medium without serum were seeded in the upper chamber of a Transwell co‐culture system (8 μm pore size; Corning Inc.) with 4 μM of aptamer, and 600 μL of medium with 10% FBS was added to the bottom chamber. After 48 hours incubation, the number of migrated cells present in the bottom chamber was counted.