Anti-bovine CD3 (Clone #MM1A, WSU, Pullman, WA) was added to 24-well plates at 10 µg/mL in 250 µL 1X PBS (Hyclone, Logan, UT), as in previous reports93 (link),94 (link). Sorted, CFSE-labeled bovine CD4+ T cells were seeded at 2 × 105 per well, and autologous neutrophils (from the same cattle) were added to the wells of T cells at either 1:1 or 10:1 (2 × 106 neutrophils) ratios. In experiments using transwell plates95 (link), CD4+ T cells were seeded in the lower chamber to interact with plate-coated anti-bovine CD3 Ab, and neutrophils were placed in a transwell insert with a pore size of 0.4 µm (Greiner Bio-one, Monroe, NC) in 100 µL medium. Neutralizing Ab against bovine IL-10 (CC320, BioRad, Hercules, CA) was added to the medium at 10 µg/mL59 (link),94 (link), and the same amount of purified mouse IgG1 (Biolegend, San Diego, CA) was supplemented in the controls. Plates were incubated at 37 °C in an atmosphere of 5% CO2 for 3.5 days and then analyzed for CFSE dilution and CD25 expression in CD4+ T cells using flow cytometry.
Transwell insert
Manufactured by Greiner
Sourced in Austria, United Kingdom, Germany, Belgium
Transwell inserts are a type of cell culture insert designed for in vitro studies of cell migration, invasion, and permeability. They consist of a porous membrane that separates two compartments, allowing the study of interactions between cells or the passage of molecules across the membrane.
Automatically generated - may contain errors
Lab products found in correlation
Matrigel, by Corning (3 mentions)
Evom2 voltohmmeter, by World Precision Instruments (3 mentions)
Matrigel, by BD (2 mentions)
Qiazol lysis reagent, by Qiagen (2 mentions)
Rsu 1 sirna, by Santa Cruz Biotechnology (2 mentions)
Collagen 1, by Corning (2 mentions)
Anti mmp13, by Abcam (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Ultroser g, by Pall Corporation (2 mentions)
Picogreen, by Thermo Fisher Scientific (2 mentions)
Polaron e3000, by Quorum Technologies (2 mentions)
Quanta 200 field emission scanning electron microscope, by Thermo Fisher Scientific (2 mentions)
Cellmask deep red plasma membrane stain, by Thermo Fisher Scientific (2 mentions)
Thincert, by Greiner (2 mentions)
Calcein am, by Merck Group (2 mentions)
Mouse igg1, by BioLegend (1 mentions)
1 25 oh 2 vitamin d3, by Merck Group (1 mentions)
β glycerophosphate β gp, by Merck Group (1 mentions)
Accuri c6, by BD (1 mentions)
62 protocols using transwell insert
1
Bovine CD4+ T Cell Activation Assay
2
Transwell Invasion Assay for SKOV3 Cells
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In preparation for the assay, a 24-well Transwell insert with 8.0 μm pore size (Greiner Bio-One, Germany) was pre-coated with 50 μg Matrigel diluted in DMEM/F12 HAM without FCS. The SKOV3 cell suspensions for monolayer and spheroid (2 × 104 cells/well in serum-free growth media + 0.1% FCS) were added to the upper compartment of the insert. Media containing a chemoattractant (10% FCS) was added to the bottom chamber of the Transwell plates. Following incubation at 37°C for 48 hours, non-invaded cells (which remained on the upper surface of the filter) were removed and invaded cells (on the lower surface of the filter) were stained with 0.5% crystal violet. The invaded cells were visualized under the microscope and counted by the image-J. The number of seeding cells was adjusted by the trypan blue (Seeding cells = 2 × 104 cells/cell viability rate).
3
Transwell Assay for NPC Cell Migration and Invasion
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NPC-039 and NPC-BM cell migration and invasion assays were performed as described by Yang et al (28 (link)). Briefly, NPC cells (3×104) were placed on the upper well of a Transwell insert (Greiner Bio-One International GmbH) with serum-free medium (RPMI-1640) and 10% FBS-containing medium (RPMI-1640 medium) (600 µl) was added to the lower chamber for 24 h at 37°C. For the invasion assay, Matrigel (25 mg/50 ml; 60 µl; BD Biosciences) was coated on the upper Transwell at 37°C, overnight. Migrated or invaded cells were fixed with 99% methanol at room temperature for 15 min and stained with Giemsa (1X) at room temperature for 2 h. Images were captured and number of cells was counted under an optical light microscope (Lecia Germany) at 100× magnification using ImageJ 1.47 version cell count software (National Institutes of Health). A total three fields of view was randomly selected for each concentration. Data are presented as the mean ± SD (n=3).
4
Phenotypic Sorting of Migratory Cancer Cells
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MDA-MB-231 breast cancer cells were phenotypically sorted based on their ability to migrate through a collagen gel on top of a Transwell insert as previously described (Hapach et al., 2021 (link)). Briefly, cancer cells were seeded on a thin layer of 1 mg/ml collagen (Corning, Corning, NY) on top of a Transwell insert with 8 μm pores (Greiner Bio-One, Kremsmunster, Austria). A serum gradient was applied and cancer cells were allowed to migrate for four days. After four days, highly migratory and weakly migratory cells were collected and reseeded in fresh Transwells. After 20 rounds of purification, cells that repeatedly migrated through the assay were termed ‘highly migratory’ (MDA+) and cells that never migrated through the assay were termed ‘weakly migratory’ (MDA-).
5
Coculture of PancTu-I cells and M2-macrophages
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For cocultures, 1 × 104 PancTu-I shCtrl or PancTu-I shTR2 cells were seeded in 2 mL/well of the respective medium into a 12-well-plate. In parallel, 2.5 × 105 M2-macrophages were seeded in 1 mL RPMI-1640 medium supplemented with 10% FCS, 1% L-glutamine (PAA) into a transwell insert (0.4 µm pore size, Greiner Bio-One, Frickenhausen, Germany), which was placed into another plate. After 24 hours, all media were exchanged and replaced by fresh PancTu-I medium and the transwells containing macrophages were transferred into wells with adherent PancTu-I shCtrl or PancTu-I shTR2 cells. In parallel, PancTu-I shCtrl or PancTu-I shTR2 cells were cultured alone under identical conditions (monoculture).
6
MSC-BV2 Crosstalk in Inflammation
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MSCs and BV2 cells were seeded and treated as described; however, after the priming treatment with IL-1, MSC spheroids were washed twice with PBS and placed into a transwell insert (0.4-μm pore size; Greiner Bio One, UK) that had been previously equilibrated in RPMI media overnight. Inserts were placed on top of BV2 cells for 24 h, after which supernatants were collected and further analysed for the presence of mouse and human cytokines by ELISA. Another set of spheroids was primed (or left unprimed) as previously described and maintained in inserts in the absence of BV2 cells. Final volumes in the wells were matched in each condition; therefore, no normalisation was carried out in this experiment. Three independent cultures were included, with at least three technical replicates per condition.
7
Generating Solid Stress in Cancer Cells
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For the generation of solid stress in cancer cells, the transmembrane pressure device was employed as previously described [70 (link)]. Briefly, cancer cells were seeded in the inner chamber of a transwell insert (Greiner Bio-one, Frickenhausen, Germany), and a 1.5 mm-thick agarose gel was added on top of the cells preventing any contact between piston and cells, providing a uniform distribution of the applied force. A piston of a predefined weight was placed on the top of the agarose gel and the cells were exposed to 4 mmHg stress. Control cells were covered with an agarose gel.
8
Osteogenic Differentiation of hASCs and hOBs under PMMA
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hASCs and hOBs were seeded into 12‐well plates at 1x104 cells/cm2, and cultured in αMEM containing 1% PSF, 10 IU/mL heparin, and 2% human PL (hASCs) or 5% human PL (hOBs) in a humidified atmosphere of 5% CO2 in air at 37°C. hASCs and hOBs were allowed to attach during 24 hr before adding PMMA material. The PMMA was placed in a transwell insert (pore size, 3.0 μm; Greiner Bio‐one, Alphen aan den Rijn, The Netherlands). Cells cultured without PMMA were used as control. After 24 h, medium was replaced with osteogenic medium containing α‐MEM (hASCs) or DMEM (hOBs), supplemented with 1% PSF, 10 IU/mL heparin, and 2% (hASCs) or 5% (hOBs) human PL, 50 μM ascorbic acid‐2‐phosphate (vitamin C; Sigma, Saint Louis, MO, USA), 5 mM β‐glycerophosphate (βGP) (Sigma) and 10 nM 1,25‐(OH)2‐vitamin D3) (Sigma). The medium was refreshed every 3 days. hASCs and hOBs were exposed to PMMA up to day 14 of culture.
9
Co-culture of PDECs and M1-Macrophages
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PDECs were seeded at 1.5 × 104 cells/well in 1 mL/well HPDE medium (50% (v/v) RPMI-1640 supplemented with 10% (v/v) fetal calf serum, 2 mM L-glutamine and 50% (v/v) keratinocyte serum-free medium supplemented with 50 µg/mL bovine pituitary extract, 5 ng/mL Epidermal Growth Factor and 4 µg/mL puromycin) into a 12-well plate. In parallel, M1-macrophages were seeded at 1.5 × 105 cell/well in 1 mL/well RPMI-1640 medium supplemented with 10% fetal calf serum and 2 mM L-glutamine into a 12-well plate transwell insert (0.4 µm pore size, Greiner, Frickenhausen, Germany). After 24 hours, media were aspirated and replaced by RPMI-1640 medium supplemented with 10% fetal calf serum and 2 mM L-glutamine. Finally, transwell inserts were placed into the wells containing adherent PDECs. Co-cultures were carried out for five days until subsequent analyses were performed.
10
Mitochondrial Membrane Potential and Cell Cytotoxicity
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The JC-1 fluorescent dye was used to measure mitochondrial membrane potential. KHYG-1 cells were treated with various doses of shuterin for 24 h at 37 °C under 5% CO2. After treatment, the cells were added to the upper wells of a Transwell insert (Greiner Bio-One, Monroe, NC, USA); K562 cells (in RPMI 1640 media) were added to the lower wells of this insert and incubated according to previously described coculture methods [58 (link)]. The effector cells were cocultured with the target cells at an effector:target (E:T) ratio of 6:1 for 2 h at 37 °C under 5% CO2. Subsequently, the K562 cells were incubated with JC-1 solution for 15 min at 37 °C. The BD Accuri C6 flow cytometer was used for data collection, and the corresponding software was used for data analysis.
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