Recombinant human ATX (also known as Ectonucleotide Pyrophosphatase/Phosphodiesterase-2) was purchased from R&D Systems, Inc., Minneapolis, MN, USA. 1-Oleoyl lysophosphatidic acid sodium salt was from Tocris Bioscience (Bristol, UK). 17-β estradiol (estrogen) was from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany). PD98059 [the phosphorylated extracellular signal-regulated kinase (p-ERK) inhibitor] was from Invitrogen (Thermo Fisher Scientific, Inc., Waltham, MA, USA). ATX antibody for western blotting was from Abcam (Cambridge, UK; ab77104) and for immunohistochemistry from Santa Cruz Biotechnology (Texas, USA; sc-374222). Antibodies against LPA receptors (LPA1, LPA2, LPA3) were from Abcam (ab166903, ab38322 and ab219267, respectively). Total/phosphorylated extracellular signal-regulated kinases (t/p-ERK) were from Cell Signaling Technology, Inc. Danvers, MA, USA (4695s and 4370s). Antibodies against GAPDH were from Santa Cruz Biotechnology (sc-51907). Crystal violet staining solution was from Tiangen Biotech Co., Ltd. (Beijing, China). Cell Counting kit (CCK)-8 was from Beyotime Institute of Biotechnology, Haimen, China (C0037). Transwell culture plates were from Corning Incorporated (Corning, NY, USA). Lipofectamine 2000™ transfection reagent was from Invitrogen, Thermo Fisher Scientific, Inc.
Transwell culture plate
Manufactured by Corning
Sourced in United States
The Transwell culture plate is a laboratory equipment product designed for cell culture applications. It features a permeable membrane support that allows for the co-culture of different cell types or the study of cell migration and transport processes. The core function of the Transwell culture plate is to facilitate the separation and interaction of cells in a controlled in vitro environment.
Automatically generated - may contain errors
Lab products found in correlation
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Cell counting kit 8 (cck8), by Beyotime (2 mentions)
Giemsa stain solution, by Solarbio (2 mentions)
Ab166903, by Abcam (1 mentions)
Ab38322, by Abcam (1 mentions)
1 oleoyl lysophosphatidic acid sodium salt, by Bio-Techne (1 mentions)
Pd98059, by Thermo Fisher Scientific (1 mentions)
Sc 51907, by Santa Cruz Biotechnology (1 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (1 mentions)
Cell counting kit 8, by Beyotime (1 mentions)
Formaldehyde solution, by Merck Group (1 mentions)
Crystal violet solution, by Merck Group (1 mentions)
Eclipse ts100, by Nikon (1 mentions)
Escherichia coli o55 b5, by Merck Group (1 mentions)
Millicell ers device, by Merck Group (1 mentions)
Caco 2 cell line, by American Type Culture Collection (1 mentions)
Biocoat matrigel invasion chamber, by BD (1 mentions)
Matrigel, by BD (1 mentions)
Sulfo nhs biotin, by Thermo Fisher Scientific (1 mentions)
Neutravidin beads, by Thermo Fisher Scientific (1 mentions)
25 protocols using transwell culture plate
1
Lysophosphatidic Acid Signaling Mechanisms
2
Evaluating MRC-5 Cell Migration
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MRC-5 migration was evaluated using 6.5 mm Transwell culture plates with an 8.0 µm pore polycarbonate membrane (Corning Incorporated, NY, USA). Cells were serum-deprived for 24 h, trypsinized and seeded in the upper chamber in serum-free culture medium supplemented with or without study compounds (10 µM). The lower compartment was filled with the same culture medium, and after 1 h TGF-β1 was added to the wells. After 24 h of incubation cells were fixed with 4% formaldehyde solution (Sigma Aldrich, St. Louis, MO, USA) and stained with 0.5% crystal violet solution (Sigma Aldrich, St. Louis, MO, USA). The non-migrated cells were removed from the upper face of the membranes using a cotton swab. The number of migrated cells was counted under an inverted microscope (Nikon Eclipse TS 100, 10× objective) using 10 randomly selected fields of view. Experiments were run twice, in a blind-folded manner.
3
Placental Bile Acid Transport Modulation
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Transwell culture plates (Corning, USA) were used to study the effects of dexamethasone on bile acid transport activity via the placenta in vitro. BeWo cells were seeded at a density of 100,000 cells/cm2 in the transwell plates. According to a previous finding [40 (link)], a confluent monolayer of BeWo cells is formed for transport experiments on day 6 post-seeding. After seeding for 24 h in the transwell inserts, the cells were treated with RU486 (10 μM) and GW4064 (10 μM), with or without dexamethasone (500 nM) for 5 days, respectively, and the culture medium (0.1 ml apical compartment, 0.6 ml basolateral compartment) was replaced daily. At day 6 post-seeding, CDCA (20 μM) and taurocholic acid (TCA, 20 μM) at a concentration that has no cytotoxicity [43 (link)] were added to the upper compartments, and the samples were collected from the upper compartments after 120 min. The concentrations of the two BA samples (CDCA and TCA) were analyzed by the TBA assay kit according to the manufacturer’s instructions.
4
Co-culture Cytokine Release Assay
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For the co-cultures, A549 cells (105 cells/well) were seeded at the bottom and THP-1 cells (105 cells/well) were plated on the insert (0.4 μm pore polyester filter) of Transwell culture plates (#3470, Corning Inc., Corning, NY, USA), with the two cell cultures being physically separated to avoid direct contact, according to the method described by Li et al. [32 (link)]. After 24 h co-culture, the cells were exposed to the following treatments: vehicle (DMSO 0.5% in PBS), CsA_rm 10 µg/mL, CsA_M20 at 10 µg/mL in respect to CsA, mannitol 2 µg/mL in DMSO 0.5% in PBS. After 1 h, LPS 1 μg/mL (Escherichia coli O55:B5; cat# L6529; Sigma Aldrich, Merck, Milano, Italy) was added to the culture and maintained for 24 h. Cells incubated with the vehicle and not exposed to LPS were used as the control. The concentration of IL-6 in the conditioned media was subsequently determined using an ELISA kit (Boster Biological Technology, Milano, Italy; cat. no. IL-6, EK0410), according to the manufacturer’s protocol and expressed as pg/mL.
5
Transwell Assay for Cell Migration
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5 × 105 cells were seeded into the upper chambers of transwell culture plates (Corning, Shanghai, China). Medium supplemented with 20% FBS (500 μl) was put into the lower chambers. H2O2 (5 μM) treated shGPX3, oeGPX3 and Ctrl cells for 4h. After washing off H2O2 with PBS, 5 × 105 cells were seeded into the upper chambers of transwell culture plates. We performed Transwell assay as above. After incubation for 24 h for migration assays, cells penetrated to the lower surface of the membrane and fixed with 4% paraformaldehyde for 60 min and then stained with crystal violet for 30 min and counted.
6
Caco-2 Cell Monolayer Transport Assay
7
Cytokine Production in HTB-5 and HMC-1 Coculture
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The production of pro- and anti-inflammatory cytokines was analyzed in cocultured HTB-5 and HMC-1 cells. Both cell types were incubated in 12-well Transwell® culture plates (Corning® Costar®, New York, NY, USA) with a permeable membrane with 4 µm pores and a culture area of 0.33 cm2. Briefly, 1 × 104 HMC-1 cells/mL were seeded in the lower chamber of the culture plate, and 9 × 104 HTB-5 cells/mL were seeded in the upper chamber. The cocultured cells were infected with UPEC strain CT073, single mutants (ΔfimH, ΔfliC, and ΔcsgA), double mutants (ΔfimHΔfliC, ΔcsgAΔfimH, and ΔcsgAΔfliC) and previously purified proteins (FimH, FliC, and CsgA) and cultured under the same conditions. At different time points after infection (3 and 5 h), the supernatants of the wells were collected and centrifuged at 500× g for 1 min. Cytokine release in the newly generated supernatants was assessed, and the pellet was discarded. PBS and culture media were used as negative controls, UPEC strain CFT073, and purified proteins (FimH, FliC and CsgA) were used as positive controls.
8
Transwell-based Cell Migration and Invasion Assay
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Transwell culture plates (Corning, UK) and BioCoat Matrigel invasion chambers (BD Biosciences) were employed to evaluate cell migration and invasion capabilities. The suspension of cells (1 × 105/mL) was made using a culture medium without FBS. Following this, 100 μL of the suspension was inoculated into the upper Transwell, while 500 μL of medium containing 20% FBS was added to the lower Transwell chamber. After incubation at 37 ℃ for 48 h, the cells in the upper chamber were removed using cotton swabs. The cells in the lower chamber were fixed using a solution of 4% paraformaldehyde and subsequently stained with crystal violet. The mean count of cells that migrated was computed by randomly selecting five fields of view under the microscope (100 ×).
9
Matrigel-based Transwell Invasion Assay
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The upper chamber of Transwell culture plates (8 µm; Corning, Inc.) was coated with 0.4 mg/ml Matrigel (BD Biosciences) and incubated for 24 h at 4°C. Cells were seeded into the upper chamber of Transwell culture plates at a density of 2×105 cells/well. The lower chamber was filled with RPMI supplemented with 10% fetal bovine serum. After 36 h, cells on the upper membrane that did not migrate were wiped with a cotton swab, and the membrane in the lower chamber was fixed with absolute ethyl alcohol and stained with 0.1% crystal violet for 30 min at room temperature. The number of migrated cells was then counted under a light microscope in at least five fields. The experiment was repeated three times with three replicates.
10
Quantifying Endothelial Angiogenesis Inhibition
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OCM-1 (1 × 105) and MUM-2B (1 × 105) cells were seeded in each well with an 8-μm pore membrane in the upper chambers of transwell culture plates (Corning, Shanghai, China). After 16 hours of incubation, the non-invading cells on the upper chamber were scraped using a cotton swab, and the membranes were then fixed with 100% methanol and stained with 0.5% crystal violet. The cells attached to the lower surface were counted and imaged under an inverted microscope (Shanghai Caikon Optical Instrument Co., Ltd., China) at × 200 magnification in nine randomly chosen fields.
For the endothelial cell tube formation assay, 55-mL growth factor-reduced Matrigel was polymerized on 96-well plates. Human microvascular endothelial cells (HMEC-1) were serum-starved in medium for 2 hours. The cells were suspended in the medium extracted from each group, then added to the Matrigel-coated wells at a density of 3 × 104 cells/well and incubated at 37°C for 24 hours. Tube formation of the endothelial cell was defined as the appearance of intraluminal space inside the tube and the quantification of anti-angiogenic activity was calculated by measuring the tube length and the number of branch points under a light microscope with an image analyzer (Confocal Quantitative Image Cytometer CQ1 software, Japan).
For the endothelial cell tube formation assay, 55-mL growth factor-reduced Matrigel was polymerized on 96-well plates. Human microvascular endothelial cells (HMEC-1) were serum-starved in medium for 2 hours. The cells were suspended in the medium extracted from each group, then added to the Matrigel-coated wells at a density of 3 × 104 cells/well and incubated at 37°C for 24 hours. Tube formation of the endothelial cell was defined as the appearance of intraluminal space inside the tube and the quantification of anti-angiogenic activity was calculated by measuring the tube length and the number of branch points under a light microscope with an image analyzer (Confocal Quantitative Image Cytometer CQ1 software, Japan).
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