The indirect coculture of CRC cells with fibroblasts was established using Transwell membranes (0.4 μm pore size, Merck Millipore) for the paracrine crosstalk study. CCD-18Co cells (5.0 × 104 cells) were seeded on the bottom of a 24-well culture plate with 500 μl fibroblast culture medium. In addition, HT-29 or Colo205 cells (1.0 × 105) were seeded onto Transwell inserts with 100 μl DMEM. After 12 h, the Transwell inserts with cancer cells were transferred to the culture plate containing fibroblasts and cocultured for 72 h. As a blank control (monoculture), fibroblast culture medium without CCD-18Co in the plate or DMEM without cancer cells in the Transwell inserts was prepared.
Transwell membrane
Manufactured by Merck Group
Sourced in United States
Transwell membranes are a type of cell culture insert used in in vitro studies. They consist of a porous membrane that separates two compartments, allowing for the study of cell migration, permeability, and co-culture experiments. The membranes are made of materials such as polyester or polycarbonate and are available in a range of pore sizes to suit different experimental requirements.
Automatically generated - may contain errors
Lab products found in correlation
Gw4869, by Selleck Chemicals (2 mentions)
Human pedf elisa kit, by BioVendor (1 mentions)
Spectramax 250 microplate reader, by Molecular Devices (1 mentions)
Transwell cell culture plate, by Corning (1 mentions)
Matrigel, by Corning (1 mentions)
Crystal violet, by Beyotime (1 mentions)
Caco 2 cells, by Merck Group (1 mentions)
Matrigel, by BD (1 mentions)
Celltiter glo 3d cell viability assay, by Promega (1 mentions)
Matrigel, by Merck Group (1 mentions)
Realtime glo annexin 5 apoptosis and necrosis assay, by Promega (1 mentions)
15 protocols using transwell membrane
1
Indirect Coculture of CRC Cells and Fibroblasts
2
Cytokine Analysis of Giardia-Infected ILCs
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ILCs (1×106 cells per well) were seeded in a 24 well culture plate with transwell membranes (Merck, USA) and incubated for 6 hr for RNA preparation and 16 hr for cytokine determination, with G. lamblia having multiplicity of infection (MOI) of 5. The culture media were collected for cytokine determination, and the ILCs were harvested for RNA preparation.
3
Co-culture of THP-1 and VSMCs
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A co-culture system of THP-1 cells and VSMCs was established using Transwell membranes (pores 0.4 μm, Merck Millipore, United States). VSMCs were plated on the bottom of a 6-well cell culture plate, THP-1 cells were seeded on the upper transwell membrane, experiments were performed after overnight culture, and VSMCs were collected separately for further manipulation.
4
Macrophage-HNSCC Crosstalk Protocol
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Macrophages were differentiated from THP‐1 cells for 48 h prior to the experiment. Macrophages (1 × 106 cells) were cocultured in a six‐well format with Transwell membranes (0.4‐μm pores, Merck Millipore, USA), whereas macrophages were placed below the membranes and the HNSCC cell lines HN6, HN6NT5EKO, HN6NT5EOE, HN6RAB27AKO, SCC25, SCC25NT5EKO, SCC25NT5EOE, SCC25RAB27AKO (1 × 105 cells) were placed on the permeable membranes. All cells were cultured with medium without FBS for 24 h and the macrophages were digested for flow cytometry analysis.
The sEVs were extracted from HN6 cells, HN6NT5EKO cells, SCC25 cells, SCC25NT5EKO cells, hBMSCs, and hBMSCNT5EOE. The sEVs (50 μg) were mixed into M2 macrophages (1 × 106 cells) and cultured for 24 h. All cells were cultured with medium without FBS.
The sEVs were extracted from HN6 cells, HN6NT5EKO cells, SCC25 cells, SCC25NT5EKO cells, hBMSCs, and hBMSCNT5EOE. The sEVs (50 μg) were mixed into M2 macrophages (1 × 106 cells) and cultured for 24 h. All cells were cultured with medium without FBS.
5
Co-culture Assay for CAFs and HNC Cells
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The co-culture assay was established using transwell membranes (pores 0.4 μm, Merck Millipore, USA) in a 12-well format. CAFs were pretreated with GW4869 (Selleck, USA; 20 μM) or DMSO for 24 h in advance. The co-culture was performed for 3–6 days whereas CAFs (5 × 104 cells) on the permeable membranes were treated with GW4869 (20 μM) or DMSO, and then HNC cells (5 × 104 cells at the beginning) below the membranes were ready for RNA extraction or further cytological experiments.
6
Co-Culture Assay with GW4869 Treatment
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The co-culture assay was established using transwell membranes (pores 0.4 μm, Merck Millipore, USA) in a 12-well format. OS cells were pretreated with GW4869 (Selleck, USA; 20 μM) or DMSO for 24 h in advance. The co-culture was performed for 3-6 days whereas OS (5 × 104 cells) on the permeable membranes were treated with GW4869 (20 μM) or DMSO, and then MRC5 cells (5 × 104 cells at the beginning) below the membranes were ready for further cytological experiments.
7
Co-culture Assay with Transwell Membranes
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The co-culture assay was established using transwell membranes (pores 0.4 μm, Merck Millipore, USA). Fibroblasts (2×104 cells in 600 μL DMEM medium containing 5% FBS) were plated on the bottom of a 24-well tissue culture plate, and 1×104 tumor cells in 300 μL DMEM medium containing 5% FBS were seeded on the transwell membranes. Experiments were performed after the cells were incubated for 72 h, and the different cell types were collected separately for further experiments.
8
PEDF Secretion in hPSC-RPE Cells
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hPSC-RPE cells were cultured on Transwell membranes (0.33 cm2, Millipore) coated with different substrates. Supernatants from both the hPSC-RPE apical and basal sides (meaning upper and lower compartments of the transwell, respectively) were collected 60 h after the medium was changed. PEDF secretion levels were measured in triplicates for each condition with commercially available human PEDF ELISA Kits (BioVendor RD191114200R) were used, in accordance with the manufacturer’s instructions, after 60 days of culture. The optical density Sreadings were measured using SpectraMax 250 Microplate Reader (Molecular Devices). Results are presented as mean ± SEM.
9
Dendritic Cell Co-culture with PIECs
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Co-cultures of cells were performed using transwell cell culture plates (Corning, Corning, NY, USA) with transwell membranes (0.1-μm pore size) (Millipore, Burlington, MA, USA). PIECs alone, PCV2-PIECs, IL-8si-PIECs, and IL-8over-PIECs were cultured in the lower chambers of the transwell plate individually at 37 °C for 24 h. MoDCs induced by the induction medium were seeded into the upper chamber of the transwell membranes. The cells were co-cultured in a humidified 5% CO2 incubator at 37 °C for 48 h. The ratio of cells in the upper chamber and lower chamber was 1:10. MoDCs co-cultured with different PIECs (DCs, PIECs-DCs, PCV2-PIECs-DCs, IL-8si-PIECs-DCs, and IL-8over-PIECs-DCs) were collected for further study.
10
Transwell Invasion Assay for SMMC-7721 Cells
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Transwell membranes (pore size 8μm, Millipore) were coated with 100 μl Matrigel (Corning; Matrigel:serum-free medium, 1:10). SMMC-7721 cells (8×104) in 200 μl serum-free medium were seeded into the upper chamber, while 900 μl medium containg with 10% FBS was added to the lower chamber. The cells were cultured for 24 h. Then, the migrated cells adhering to the lower surface were fixed with 4% paraformaldehyde and stained with 0.5% crystal violet dye. The number of cells were counted in 10 randomly selected visual fields viewed under a microscope.
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