Transwell cell culture inserts

Manufactured by Merck Group

Sourced in United States

Transwell cell culture inserts are a laboratory equipment used for in vitro cell culture studies. They consist of a permeable membrane insert that fits into a well of a cell culture plate, creating a dual-chamber system. This allows for the study of cell-cell interactions, transport processes, and other biological phenomena by separating cell populations while maintaining communication between them.

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Lab products found in correlation

Calcitriol, by Merck Group (1 mentions) Kgm gold medium, by Lonza (1 mentions) Ll 37, by InvivoGen (1 mentions) Dakotm fluorescent mounting medium, by Agilent Technologies (1 mentions) Basic fibroblast growth factor (bfgf), by Thermo Fisher Scientific (1 mentions) U251 cells, by American Type Culture Collection (1 mentions) Penicillin, by Thermo Fisher Scientific (1 mentions) Streptomycin, by Thermo Fisher Scientific (1 mentions) Annexin 5 cy5, by BD (1 mentions) 4 6 diamidino 2 phenylindole dihydrochloride dapi, by Beyotime (1 mentions) Trizol, by Thermo Fisher Scientific (1 mentions) Calcein am, by Thermo Fisher Scientific (1 mentions) Matrigel, by BD (1 mentions) Bradford protein assay kit, by Beyotime (1 mentions) Nuclear and cytoplasmic protein extraction kit, by Beyotime (1 mentions) Cell counting kit 8, by Beyotime (1 mentions) Ouabain, by MedChemExpress (1 mentions) Encorafenib, by MedChemExpress (1 mentions) Paraformaldehyde fix solution, by Beyotime (1 mentions) Annexin 5 pi double staining, by BD (1 mentions)

14 protocols using transwell cell culture inserts

Coculture of MSCs, EPCs for Regenerative Studies

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For all subsequent experiments, cells between passages 2 and 3 were used. MSCs, depleted-MSCs, and EPCs (CD34+/CD133+) were enzymatically detached (Trypsin-EDTA), counted, seeded at a density of 5,000 cells/cm² in 3 different culture setups (Figure 1), and incubated for 3, 7, or 10 days in presence of IMDM-FCS (IMDM supplemented with 10% FCS and 1% NEAA) or IMDM-PL (IMDM supplemented with 5% FCS, 5% PL, and 1% NEAA).
For indirect cocultures (transwell), MSCs (or depleted-MSCs) were seeded in the bottom part of 6-well plates at a density of 5,000 cells/cm² for transwell culture setup (Figure 1(a)), whereas EPCs were seeded at the same density in the corresponding transwell cell culture inserts (0.4 μm pore size, Sigma-Aldrich).
For direct coculture experiments (direct coculture), MSCs and EPCs, as well as depleted-MSCs and EPCs, were coseeded in a 1 : 1 ratio in 75 cm² cell culture plate (BD Biosciences) at an initial density of 5,000 cells/cm² (Figure 1(b)). In addition, each cell type was seeded individually in 6-well plates (5,000 cells/cm²) for single culture as controls (Figure 1(c)).

Loibl M., Binder A., Herrmann M., Duttenhoefer F., Richards R.G., Nerlich M., Alini M, & Verrier S. (2014). Direct Cell-Cell Contact between Mesenchymal Stem Cells and Endothelial Progenitor Cells Induces a Pericyte-Like Phenotype In Vitro. BioMed Research International, 2014, 395781.

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Cultivating 3D Human Airway Tissues

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EpiAirway™ 3D human respiratory epithelial tissues (AIR-100 PC6.5/PE6.5) were obtained from MatTek Corporation. The tissues were cultivated at the air–liquid interface and medium (MatTek Corporation) changed every other day according to the instructions of the producer. EpiAlveolar™ 3D tissues with and without THP-1 macrophages (MatTek Cooperation) were maintained at the air–liquid interface with medium (MatTek Corporation) changes every other day. Since EpiAlveolar™ 3D tissues with THP-1 macrophages did not tolerate the long delivery, they were added to the EpiAlveolar™ 3D tissues in the laboratory in Graz following the protocol of the company in which 25,000 THP-1 macrophages were added per insert. The cells were seeded in 75 µL media into the apical compartment during feeding and after 24 h the culture was switched back to air-liquid interface condition.
MucilAir™ tissues were purchased from Epithelix Sárl (Geneva, Switzerland) and maintained in MucilAir™ culture medium (Epithelix Sárl) in 24-well format Transwell^® cell culture inserts (Sigma-Aldrich) in a humidified incubator (37 °C; 5% v/v CO₂). The culture medium was changed every 2–3 days.

Meindl C., Absenger-Novak M., Jeitler R., Roblegg E, & Fröhlich E. (2023). Assessment of Carbon Nanotubes on Barrier Function, Ciliary Beating Frequency and Cytokine Release in In Vitro Models of the Respiratory Tract. Nanomaterials, 13(4), 682.

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Skin Bioassay for Ex Vivo Topical Treatments

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Healthy human skin samples received at Nestlé Skin Health R&D are derived from abdominoplasty surgery and obtained with the patient’s consent and with local IRP approval. Skin biopsies were performed using 8-mm biopsy punches and placed on Transwell cell culture inserts, 24 mm diameter with a 8-µm polycarbonate membrane (Costar; Sigma Aldrich) in 6-well plates containing KGM-Gold medium (Lonza) with growth supplements. For the experiment (n = 3 replicates/condition), surgical steel rings were deposited on the surface of the skin to allow topical treatment. Compounds were applied on skin ex vivo at 1 µl/0.1 cm². After overnight incubation, a second treatment with the same compound was applied 1 h before skin stimulation either by topical treatment with calcitriol (1α,25-dihydroxyvitamin D3; Sigma Aldrich) or with LL-37 (Invivogen). Biopsies were allowed to incubate for up to 72 h. In order to proceed to histologic analysis on each replicate of treatment, a biopsy of 4 mm in diameter was performed inside the surgical steel rings. The sample was then tape-stripped ten times using D-squames discs (Monaderm, Monaco) and stored in 2-mL microtubes at −80 °C until protein analysis. Following tape-stripping, further 4-mm biopsies were carried out in order to perform gene expression and protein analysis.

Thibaut de Ménonville S., Rosignoli C., Soares E., Roquet M., Bertino B., Chappuis J.P., Defoin-Platel/Chaussade C, & Piwnica D. (2017). Topical Treatment of Rosacea with Ivermectin Inhibits Gene Expression of Cathelicidin Innate Immune Mediators, LL-37 and KLK5, in Reconstructed and Ex Vivo Skin Models. Dermatology and Therapy, 7(2), 213-225.

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Transwell Migration Assay Protocol

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A chamber of non-type I-collagen- coated 24-well Transwell cell culture inserts (EMD Millipore) was used for the Transwell migration assay. Briefly, after infection with or without adenoviruses and recombination proteins for various durations, the cells were trypsinized, washed, and suspended in 400 μl of serum-free DMEM and finally seeded in the upper chamber (HeLa, 4×10⁴ cells per well; SiHa, 6×10⁴ cells per well). The lower chamber was coated with 700 μl of DMEM containing 20% FBS as a chemoattractant. After 24 h, cells were fixed with methanol for 20 min and stained with 0.05% crystal violet for 30 min at room temperature. Cells on the upper surface of the insert membrane were removed with cotton swabs. Finally, the cells were counted using an inverted microscope (magnification, ×100) in five randomly selected fields for each well. The experiment was repeated three times.

Zha H., Li X., Sun H., Duan L., Yuan S., Li H., Li A., Gu Y., Zhao J., Xie J, & Zhou L. (2019). S100A9 promotes the proliferation and migration of cervical cancer cells by inducing epithelial-mesenchymal transition and activating the Wnt/β-catenin pathway. International Journal of Oncology, 55(1), 35-44.

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BV-2 Cell Migration Assay

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BV-2 cells were plated in transwell cell culture inserts (8.0 µm pore diameter, Merck Millipore, Burlington, MA, USA) at a density of 5 × 10⁴ cells/cm², and then immediately challenged with EHP for 4 h. At the end of the experiment, the cells in the upper side of the transwell were removed with a cotton swab, and the cells at the bottom side of the insert (cells that migrated) were fixed with 4% PFA with 4% sucrose for 10 min. Nuclei were stained with DAPI (1:2000) to allow cell counting. The membrane was removed from the insert and mounted with Dako^TM Fluorescent Mounting Medium (Agilent, Santa Clara, CA, USA) in glass slides. The samples were observed in an inverted fluorescence microscope and eight fields were randomly acquired from each condition. The number of migrated cells was counted.

Ferreira-Silva J., Aires I.D., Boia R., Ambrósio A.F, & Santiago A.R. (2020). Activation of Adenosine A3 Receptor Inhibits Microglia Reactivity Elicited by Elevated Pressure. International Journal of Molecular Sciences, 21(19), 7218.

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Transwell Assay for Cell Migration

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BV-2 cells were cultured in serum-free medium for 24 h before the experiment. Then, cells were plated in transwell cell culture inserts (8.0 μm pore diameter; Merck Millipore) in RPMI with 2% FBS and 1% antibiotics. Cells were incubated with apyrase (30 U/mL) and ADA (1 U/mL) followed by exposure to EHP for 4 h. At the end of the experiment, cells were washed with warm PBS and the cells in upper side of the insert were removed with a cotton swab. After fixation with 4% PFA with 4% sucrose during 10 min, the nuclei were stained with DAPI (1:2,000). The membranes were mounted in glass slides with glycergel mounting medium, and the preparations were observed with a fluorescence microscope (Axio Observer.Z1) using a N-Achroplan 5x/0.15 M27 objective. Five random images per sample were acquired. The number of cells in the bottom side of the insert (the cells that migrated) was counted and the results were expressed as percentage of control.

Rodrigues-Neves A.C., Aires I.D., Vindeirinho J., Boia R., Madeira M.H., Gonçalves F.Q., Cunha R.A., Santos P.F., Ambrósio A.F, & Santiago A.R. (2018). Elevated Pressure Changes the Purinergic System of Microglial Cells. Frontiers in Pharmacology, 9, 16.

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Tumor-Endothelial Cell Co-Culture Protocol

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GL261 (mouse glioma cell line) and b.END3 (mouse brain microvessel endothelial cell line) cells were purchased from the Cell Bank of Shanghai Institute of Cell Biology and Biochemistry, Chinese Academy of Sciences (Shanghai, China). U251 cells (human GBM cell line) and human umbilical vein endothelial cells (HUVECs) were purchased from ATCC (Manassas, VA, USA). Mouse cells were cultured as previously described 19 (link). Human cells were cultured using Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS). Tumour spheres were cultured in serum-free DMEM/F12 medium with 10 ng/ml epidermal growth factor (EGF, PeproTech), 10 ng/ml bFGF (PeproTech) and 2 mg/ml B27 (Sigma) (stem cell medium). For co-culture experiments, transwell cell culture inserts (0.4 µm pore size; Millipore, USA) were used. Briefly, 2 × 10⁵ tumour cells with 2.6 ml medium were seeded in the lower chamber of six-well plates, and 1 × 10⁵ ECs with 1.4 ml medium were seeded in the upper chamber of the insert. After 24 h of culture, the media were changed and the insert was transferred into the well with tumour cells. After co-culture for an additional 48 h, the cells were harvested.

Yan G.N., Yang L., Lv Y.F., Shi Y., Shen L.L., Yao X.H., Guo Q.N., Zhang P., Cui Y.H., Zhang X., Bian X.W, & Guo D.Y. (2014). Endothelial cells promote stem-like phenotype of glioma cells through activating the Hedgehog pathway. The Journal of Pathology, 234(1), 11-22.

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Cell Culture and Characterization Protocol

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Cell culture medium L15, penicillin and streptomycin were purchased from Gibco (Grand Island, NY, USA). Bradford protein assay kit, cell counting kit (CCK-8), rabbit anti-human p65 polyclonal antibody, Cy3-labeled goat anti-rabbit secondary polyclonal antibody, 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI), nuclear and cytoplasmic protein extraction kit, and biotin-labeled NF-κB oligonucleotides were obtained from Beyotime Institute of Biotechnology (Jiangsu, China). Fetal bovine serum (FBS) was from HyClone (Logan, Utah, USA). Matrigel and Annexin V-cy5 were purchased from BD Biosciences (CA, USA). Trizol and Calcein-AM were from Invitrogen (Carlsbad, CA, USA), and Transwell cell culture inserts were from Millipore (MA, USA). All other chemicals were from Sigma-Aldrich (St. Louis, MO, USA) unless specified otherwise.

Li L., Zhao F., Lu J., Li T., Yang H., Wu C, & Liu Y. (2014). Notch-1 Signaling Promotes the Malignant Features of Human Breast Cancer through NF-κB Activation. PLoS ONE, 9(4), e95912.

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Hepatocyte Apoptosis Activates Hepatic Stellate Cells

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HepG2 (from Stem Cell Bank of the Chinese Academy of Sciences, Shanghai, China) and HSC-T6 (Central South University, Changsha, China) were cultured in Dulbecco׳s modified Eagle׳s medium containing 10% fetal calf serum.
A modified co-culture model²⁰ (link) was enrolled to investigate the effect of hepatocellular apoptosis on HSC activation. Briefly, HSC-T6 cells were plated in 12-well plates, while HepG2 cells were plated on permeable polycarbonate inserts (Transwell cell culture inserts, Millipore, USA) in another 12-well plate. HepG2 cells were incubated with CHX (40 μmol/L) for 30 min, followed by the addition of TNFα (20 ng/mL) for 12 h to induce apoptosis as previously described²² (link). Then the cell-culture inserts containing apoptotic HepG2 cells were transferred onto the HSC-T6 cells after washed and new medium was added.

Zhou J., Huang N., Guo Y., Cui S., Ge C., He Q., Pan X., Wang G., Wang H, & Hao H. (2018). Combined obeticholic acid and apoptosis inhibitor treatment alleviates liver fibrosis. Acta Pharmaceutica Sinica. B, 9(3), 526-536.

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Investigating Cytotoxic Effects of Ouabain and Encorafenib

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The chemicals ouabain and encorafenib were purchased from MedChem Express (Princeton, NJ, USA). Cell counting kit-8 (CCK8), crystal violet staining solution, 4% paraformaldehyde fix solution, and cell cycle and apoptosis analysis kit were purchased from Beyotime Biotechnology (Shanghai, China). The apoptosis detection kit (Annexin V-PI double staining) was purchased from BD Biosciences (San Jose, CA, USA). Transwell cell culture inserts with 8 μM diameter were purchased from Millipore Corporation (Bedford, MA, USA). The antibodies against Bcl-2, Bax, and β-actin were purchased from Cell Signaling Technology (Beverly, MA, USA).

Wang L., Cai W., Han B., Zhang J., Yu B, & Chen M. (2021). Ouabain Exhibited Strong Anticancer Effects in Melanoma Cells via Induction of Apoptosis, G2/M Phase Arrest, and Migration Inhibition. OncoTargets and therapy, 14, 1261-1273.

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