Caco-2 cells (106 cells) were added to inserts (HTS Transwell 96-well; Corning; 3387) in the presence of Dulbecco’s Modified Eagle Medium (DMEM) for 24 h [25 (link)]. Dextran sulfate sodium (DSS; 1.5%; MP Biomedicals, LLC, Eschwege, Germany) was then added to each well for 24 h. Caco-2 cells were then treated with FA at a concentration of 25 µg/mL. In parallel, THP-1 cells were differentiated into macrophages using PMA at a concentration of 200 ng/mL for 72 h. After different washings with PBS, the macrophages were maintained in RPMI for 24 h. They were then labeled with fluorescent calcein (Invitrogen; Villebon sur Yvette, France) and added to each insert at a concentration of 105 macrophages/insert. RPMI (150 µL) containing 105 C. albicans yeast cells was then added to the lower chamber, and the plates were incubated at 37 °C in 5% CO2 overnight. After overnight migration, the upper chamber of the Transwell inserts containing nonmigrated macrophages was removed from the plate, and migrated macrophages present on C. albicans cells were determined by measuring fluorescence using a fluorometer (FLUOstar® Omega; BMG Labtech, Saitama, Japan). Macrophage migration through Caco-2 cells untreated with DSS was assigned a value of 100%, corresponding to a healthy intestinal barrier, while the value 200% corresponded to a destroyed intestinal barrier.
96 well hts transwell
Manufactured by Corning
Sourced in United States
The 96-well HTS Transwell is a laboratory equipment designed for high-throughput screening applications. It provides a standardized platform for performing cell-based assays that require the separation of cell populations while allowing for the exchange of media and other substances between the upper and lower chambers.
Automatically generated - may contain errors
Lab products found in correlation
Fluostar omega, by BMG Labtech (1 mentions)
Dextran sulfate sodium (dss), by MP Biomedicals (1 mentions)
Recombinant human mmp 9 protein, by Abcam (1 mentions)
96 well woundmaker, by Sartorius (1 mentions)
Essen imagelock microplate, by Sartorius (1 mentions)
Tyrphostin ag490, by Merck Group (1 mentions)
Incucyte s3 live cell analysis system, by Sartorius (1 mentions)
Biocoat matrigel invasion chamber, by BD (1 mentions)
5 protocols using 96 well hts transwell
1
Macrophage Migration Across Inflamed Intestinal Barrier
2
In Vitro Wound Healing Assay with Fibroblast-MNC Coculture
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In 96-well Essen ImageLock microplates (Essen BioScience, Ann Arbor, MI, USA), fibroblasts were attached with DMEM containing 10% FBS. Then, they were cultured under the abovementioned serum starvation condition. Furthermore, we created wounds by using a 96-well wound maker (Essen BioScience). After removing the detached cells, the wells were added with culture inserts (HTS Transwell 96-Well; catalog No.7369; Corning, NY, USA) containing an equal number of MNCs, MNC-QQc cells, or recombinant human MMP9 protein (500 pg/mL) (Abcam, Cambridge, UK), with or without MMP9 inhibitor 1 (1 μM) (Merck Biosciences, Darmstadt, Germany) or tyrphostin AG 490 (40 μM) (Sigma–Aldrich, St. Louis, MO). The culture insert is a porous membrane made of 0.4 μm polyethylene terephthalate material that allows the easy permeation of cytokines. The culture insert has a suspended design, which allows it to hang in the middle of the well. Fibroblasts were attached to the bottom of the plate well, and MNCs or MNC-QQc cells were seeded in the insert wells. Two types of cells were co-cultured in a non-contact manner using the culture insert device, and only the medium was common. Then, the fibroblasts on each well were imaged using an IncuCyte S3 Live-cell analysis system (Essen BioScience, MI, USA), and the devoid area of cells in the images was measured using the ImageJ software.
3
Evaluating Anti-Cancer Drug Effect on Cell Migration and Invasion
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Cell migration assay was performed in Corning’s 96-well HTS Transwell as per manufacturer’s instructions. Cells were treated with VERU-111 (1.25–10 nM) for 24 h, fixed with 4% para-formaldehyde, and with stained with crystal violet. Further, a wound healing assay was also performed to evaluate the effect of VERU-111 on cell migration. Images of the wounds were monitored under a phase-contrast microscope at 10X magnification. For Invasion assays, cells were grown on BD Biocoat Matrigel Invasion Chambers (BD Biosciences, Heidelberg, Germany) according to the manufacturer’s protocol. Cells were then treated with different concentrations of VERU-111 and incubated for 24 h. Invaded cells were fixed, stained and counted as described in our previous study [30 (link)].
4
Transwell Assay for Cell Migration
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Cell migration was carried out as per the instructions of the manufacturer in Corning’s 96-well HTS trans-well. PanCa cells (AsPC-1 and HPAF-II) were seeded at a density of 50,000 cell/well in the upper chamber of the plate containing serum-free culture medium and then treated with Cuc D for 18 h, following which cells were allowed to migrate to lower chamber containing 10% FBS. After the completion of time, the cells in the upper chamber were completely removed by cotton swab, and the cells in the lower chamber were fixed in 4% paraformaldehyde solution. These cells were further stained with crystal violet. A phase contrast microscope was used to observe the migrated cells.
5
Transwell Cell Migration Assay
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Cell migration assay was performed in Corning’s 96-well HTS transwell as per manufacturer’s instructions with minor modifications. Briefly, Caski cells (50,000 cells/well) were seeded in upper chambers of the plate containing serum-free culture medium. Cells were further treated with BaP alone, or in combination (BaP and CUR/Nano-CUR) for 18 hrs. Cells were allowed to migrate from upper chamber (5% FBS) towards lower chamber which has 10% FBS. Cells in the upper chamber were completely removed by cotton swab. Cells were fixed with 4% paraformaldehyde prepared in PBS for 30 min. These cells were stained with Giemsa stain and migrated cells were observed by using a light microscope. Migrated cells were counted in six random fields of view and the experiments were performed in triplicate.
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