Transwell cell culture inserts (Greiner Bio‐One) with 3 μm pore‐size filters were placed in individual wells of 24‐well plates. Briefly, 4B12 osteoclastogenic precursor cells (5 × 104 cells/well) were seeded and grown on the well plates; subsequently, pre‐osteoblastic MC3T3‐E1 cells (3 × 104 cells/mL) were seeded and grown on the transwell inserts on top. Pre‐osteoblasts were then supplemented with the two concentrations of extract from Mn(II)‐enriched C glomerate and challenged with LPS for osteoclastogenic factors production. Cocultures were thus maintained for 10 days in standard culture conditions. On the 11th day, inserts were discarded, and osteoclastogenic‐4B12 differentiated cells were collected for TRAP staining, RT‐PCR analysis and Phalloidin labelling as previously described for confocal evaluation.
Transwell cell culture inserts
Manufactured by Greiner
Sourced in United States, Austria
Transwell cell culture inserts are a type of laboratory equipment used for cell culture applications. They consist of a porous membrane that allows for the separation of cells or media between the upper and lower chambers of a multi-well culture plate. This design enables researchers to study cell migration, permeability, and other cellular processes in a controlled environment.
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4 protocols using transwell cell culture inserts
1
Osteoclastogenic Effects of Mn(II)-enriched C. glomerata
2
Transwell Assay for Cell Migration
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Metastatic and primary C26.WT cells were seeded on three collagen (Sigma-Aldrich; Merck KGaA) precoated transwell cell culture inserts (Greiner Bio-One North America Inc., Monroe, NC, USA) in 24-well culture plates with RPMI-1640 without serum in the upper chamber at a concentration of 2×105 cells/ml, and RPMI-1640 with 0.5% FBS (Gibco; Thermo Fisher Scientific Inc.) was plated in the lower chamber. Following 18 h of incubation, the migration levels were assessed by staining the chambers with eosin at room temperature for 10 min. The number of migrated cells was estimated under a light microscope (magnification, ×20) using ImageJ 1.48v software (National Institutes of Health, Bethesda, MD, USA). The experiment was repeated three times.
3
Co-culture Transwell Assay Protocol
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Co‐culture experiments were performed using Transwell cell culture inserts (Greiner Bio‐One, Monroe, North Carolina) in 6‐well or 24‐well plates. Briefly, cells were added to the lower compartment and allowed to attach for 12‐24 hours. For the migration assay, cells were placed into the upper compartment, the reagents were added to the lower compartment and the plates were cultured for 24‐48 hours. For the proliferation assay, cells were placed into the lower compartment and allowed to attach for 12‐24 hours. Co‐cultured cells were then added to the upper compartment and the plates were cultured for 24‐72 hours.
4
Collagenase-Mediated Protein Release Assay
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The constructs were prepared (n = 3 per sample) in Transwell cell culture inserts (Greiner Bio-One, Kremsmünster, Austria) and incubated in 0.0015% type I collagenase (2 U/mL) (Whartington, UK) at 37 °C for 24 h according to the protocol described by Sakamoto et al. [28 (link)]. A total 200 µL of the released protein in collagenase solution was sampled for the first 30 min and at subsequent 2 h intervals for 24 h (n = 3 each time). The solutions were kept at −80 °C until they were measured using the BCA assay, with the absorbance read at 562 nm.
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