24 well cell culture insert

Migration and invasion assays were performed in 24‑well cell culture inserts (Corning) fitted with a PET membrane (8-μm pore size). The inserts for invasion assays were coated with 30 μL Matrigel matrix at 37 °C for 1 h. Transfected cells were plated in medium without serum in the top chamber of a transwell. The bottom chamber contained 600 μL RPMI 1640 medium with 10% FBS. After incubating for 24 h at 37 °C, the cells that had migrated to the lower surface of the membrane were fixed with 4% methanol, stained with crystal violet and photographed under a microscope. Cell numbers were counted under a light microscope at 200 × magnification.

Human colonoids were prepared from biopsy samples of adult healthy individuals at the Digestive Diseases Research Center of the Washington University, School of Medicine and were grown as previously described (49 (link), 50 (link)). Briefly, isolated colonoids were thawed and plated (in 24-well plates) in Matrigel (BD Biosciences) droplets (15 μl) followed by incubation at 37 °C with conditioned media (a 1:1 mixture of the L-WRN cell line and primary culture media [Advanced DMEM/F12; Invitrogen] supplemented with fetal bovine serum [20%], l-glutamine [2 mM], penicillin [100 U/ml], streptomycin [100 μg/ml], Stemolecule Y27632 [10 μM; Reprocell], and SB 431542 [10 μM; Peprotech]). To obtain polarized differentiated colonoid monolayers, cells were plated at density of 5 × 10⁴ cells/well onto 24-well cell culture inserts (Corning) coated with type IV human collagen (Sigma) and then grown for 4 days prior to induction of differentiation by addition of differentiation media (5% conditioned media added with only Y-27632 inhibitor). Differentiated colonoid monolayers were then exposed as indicated to normoxia and hypoxia conditions as mentioned previously.

Cell invasion was assessed using transwell cell culture chambers, according to the manufacturer's protocol. We used 24‐well cell culture inserts (Corning, USA) with a polyethylene terephthalate membrane (8 μM pores). The membranes of each upper chamber were coated with Matrigel (100 μg/cm²; BD) and then incubated at 37°C overnight for gelling. Before each assay, the WRO 82‐1 cells were treated with nevirapine at the indicated concentration for 72 hours. For cell invasion assay, 200 μL of WRO 82‐1 cells (5 × 10⁵/mL in serum‐free medium) were seeded in the upper chamber in serum‐free medium, and 750 μL of media supplemented with 10% FBS was added to the lower chamber. The cells were then incubated at 37°C for 48 hours, washed twice with PBS, fixed with 3.7% formaldehyde at room temperature for two minutes, permeabilized by 100% methanol at room temperature for 20 minutes, and then stained with crystal violet for 15 minutes at room temperature. The cells that adhered to the upper surface of the chamber were carefully removed using cotton swabs, and those on the bottom surface of the membrane were imaged, after which cells in five randomly selected fields were counted under a light microscope (Leica, Germany) at 20× objective magnification. Experiments were performed in triplicate.

Caco-2 cells (human colorectal adenocarcinoma cell line; CLS, Germany) were maintained in the Caco-2 medium. The cells were seeded at a density of 2 × 10⁴ cells/cm² on 24-well cell culture inserts (Corning, USA) and maintained for 21 days to reach a differentiated monolayer. The apical (300 μl) and basolateral (800 μl) culture media were changed three times per week.
HaCaT cells (human keratinocyte cell line; CLS, Germany) were maintained in HaCaT medium. The cells were seeded at a density of 4 × 10⁴ cells/cm² on a 24-well or 96-well plate (TPP, Switzerland) and maintained for 3 days to reach a confluence.

2.5 × 10⁴ hepatoma cells were resuspended in 100 μL medium containing 1 % FCS and transferred onto 24-well cell culture inserts with a pore size of 8 μm (Corning, Tewksbury, USA). 600 μL medium containing 10 % FCS was added to lower chambers to generate a gradient for cell migration. Medium was removed and migrated cells on membranes were fixed with 4 % paraformaldehyde after 16 h. Cell nuclei of migrated cells at the bottom side of the membrane were stained with Hoechst 33342 (Life Technologies, Green Island, USA) and counted under the fluorescence microscope (Nikon, Tokyo, Japan). To analyze cell invasion, 24-well cell culture inserts were coated with rat-tail collagen (BD Biosciences, NJ, USA) prior to seeding of hepatoma cells. To examine transendothelial invasion, 2 × 10⁵ HSECs were plated in 100 μL endothelial cell medium (Lonza, Basel, Switzerland) onto coated 24-well cell culture inserts and allowed to form a monolayer for 48 h. Subsequently, endothelial cell medium was aspirated and hepatoma cells were seeded in 100 μL medium containing 1 % FCS onto 24-well inserts. Transmigrated cells were visualized after 16 h and quantified as described for cell migration.