Upper inserts

Manufactured by Corning

Sourced in United States

The Upper Inserts are a component of laboratory equipment designed to provide a secure and stable platform for various experimental setups. They are made of durable materials and engineered to offer a reliable and consistent solution for laboratory researchers and technicians.

Automatically generated - may contain errors

Lab products found in correlation

Trizol reagent, by Thermo Fisher Scientific (2 mentions) 24 well transwell plate, by Corning (1 mentions) Hk 2 cells, by Merck Group (1 mentions) Human angiotensin 2 ang 2, by Merck Group (1 mentions) Thp 1 cells, by RIKEN BioResource Center (1 mentions) Recombinant human tgf β1, by R&D Systems (1 mentions) Ripa buffer, by Merck Group (1 mentions) Phorbol 12 myristate 13 acetate, by Merck Group (1 mentions) Tgf β1, by R&D Systems (1 mentions) Hk 2 cell, by American Type Culture Collection (1 mentions) 24 well plate, by Corning (1 mentions) Paraformaldehyde pfa, by Wuhan Servicebio Technology (1 mentions) Dm2000, by Leica (1 mentions) Bx 51 dp 72, by Olympus (1 mentions)

5 protocols using upper inserts

Cell and Monocyte Migration Assays

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For the cell migration assay, cells (5 × 10⁴ per well) with 1% FBS were seeded in the upper inserts (Corning, NY, USA) and then exposed to media consisting of 10% FBS or the indicated CM in the lower chambers (inserts with 8-μm pore size). Cells migrating to the lower surface of the inserts were counted after 12 h and analyzed using Image-Pro Plus 6.0 software.
For the monocyte chemotaxis assay, purified monocytes (1 × 10⁶ per well) were seeded in the upper chambers of a 24-well Transwell plate (Corning, NY, USA) and exposed to media containing the indicated CMs in the lower chambers (inserts with 0.4-μm pore size) for 12 h. The chemotaxis of monocytes was analyzed by counting the number of migrated cells.

Tang J., He J., Guo H., Lin H., Li M., Yang T., Wang H.Y., Li D., Liu J., Li L., Xia H., Zhuo Z, & Miao L. (2023). PTBP2-Mediated Alternative Splicing of IRF9 Controls Tumor-Associated Monocyte/Macrophage Chemotaxis and Repolarization in Neuroblastoma Progression. Research, 6, 0033.

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Mesenchymal Stem Cell Modulation of Macrophage Phenotype

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HK‐2 cells (American Type Culture Collection, Manassas, VA) and THP‐1 cells (Riken BRC) were maintained as described previously 38, 39. After the starvation of HK‐2 cells with SF‐hMSC‐CM (with or without TSG‐6 siRNA transfection), 10%hMSC‐CM, or serum‐free DMEM for 24 hours, the cells were exposed to 1 μM human angiotensin‐II (Ang‐II; Sigma–Aldrich) for 12 hours, 5 ng/ml recombinant human TGF‐β1 (R&D Systems, Minneapolis, MN) for 12 hours, or 10 ng/ml TGF‐β1 for 1 hours or 48 hours. Whole cell lysates were prepared in RIPA buffer (Sigma–Aldrich) and analyzed. Total RNA was extracted using TRIzol Reagent (Thermo Fisher Scientific) and subjected to PCR.
THP‐1 cells were differentiated into M1 macrophages by treatment with phorbol 12‐myristate 13‐acetate (160 nM; Sigma–Aldrich) for 48 hours and then stimulated for a further 24 hours with recombinant human interferon‐γ (20 ng/ml; Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). A Transwell cell coculture system (Corning, Corning, NY) was used to examine the phenotypic change of macrophages by MSCs without direct cell contact. Upper inserts (pore size, 1.0 μm; Corning) with cultured SF‐hMSCs, 10%hMSCs, or SF‐hMSCs transfected with TSG‐6 siRNA (5 × 10⁴ cells/insert) were dipped into the basal plate of cultured THP‐1 macrophages. After 48 hours, the surface antigens of THP‐1 macrophages were determined by flow cytometry.

Yoshida K., Nakashima A., Doi S., Ueno T., Okubo T., Kawano K., Kanawa M., Kato Y., Higashi Y, & Masaki T. (2018). Serum‐Free Medium Enhances the Immunosuppressive and Antifibrotic Abilities of Mesenchymal Stem Cells Utilized in Experimental Renal Fibrosis. Stem Cells Translational Medicine, 7(12), 893-905.

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Astrocyte-Microglia Co-culture Assay

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As shown in Fig. 4A, the primary astrocytes were seeded into 24-well plates (Corning, Corning, NY, USA), and microglia undergoing specific treatment in each group were inoculated in the upper inserts (Corning). After 48 h incubation, the medium and astrocytes were collected for subsequent experiments.

Qian Z., Chen H., Xia M., Chang J., Li X., Ye S., Wu S., Jiang S., Bao J., Wang B., Kong R., Zhang S., Zheng S., Cao X, & Hong X. (2022). Activation of glucagon-like peptide-1 receptor in microglia attenuates neuroinflammation-induced glial scarring via rescuing Arf and Rho GAP adapter protein 3 expressions after nerve injury. International Journal of Biological Sciences, 18(4), 1328-1346.

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Astrocyte Migration in Microglia Co-culture

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Primary astrocytes (1 × 10⁵) were resuspended in 200 μL FBS-free DMEM and seeded into the upper inserts (Corning). Media with microglia (MG) in each group was collected after 24 h of culture, then added in a volume ratio of 1:1 with complete DMEM in each of the lower layers, which underwent 24 h of culture. The astrocytes in the upper inserts were fixed with 4% paraformaldehyde (PFA; Servicebio, Wuhan, China) followed by staining with Crystal Violet reagent (KeyGen). We observed cells under the permeable membrane using a microscope (DM2000, Leica, Wetzlar, Germany).

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Evaluating Cell Invasion with Transwell Assay

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Cell invasion was evaluated by transwell chamber assay as previously described [19] . Brie y, the upper inserts (8 µm pore size, 6.5 mm diameter, Corning, USA) pre-coated with 200 µl matrigel were placed at 37 °C for 1 h in a CO 2 incubator. Both hESC-EVT and hiPSC-EVT (2.5 × 10 4 cells/well) cells were treated with various concentrations of CsA (0, 1.0, and 10 uM, respectively) in the upper chamber after the matrigel were removed. The lower chamber was added with 600 µL EVT basal medium supplemented with 10% FBS. Cells were cultured in 5% CO2 and 37 °C for 48 h. The cells without invading the lters were removed with a cotton swab from the upper surfaces. The inserts were xed for 30 min with 4% paraformaldehyde, and then stained with Giemsa Stain solution at room temperature for 10 min. Staining was captured with Olympus microscope (BX51 + DP72, Olympus, Japan). The cells invaded the lters to the lower surfaces were counted in 6 randomly selected at a magni cation of × 400, and non-overlapping elds. Each experiment was performed in triplicate, and duplicated at least 3 times. The migration index was determined by the rate of the percentage of cell invasion from the varied concentrations of CsA to that of the vehicle.

Wang J., Long P., Tian S., Zu W., Liu J., Wu B., Mao J., Li D., Ma Y, & Huang Y. (2023). Cyclosporin A Promotes Invasion and Migration of Extravillous Trophoblast Cells Derived from Human-Induced Pluripotent Stem Cells and Human Embryonic Stem Cells. Stem cells and development, 32(3-4).

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