A solution of NP(pIC+R848+MIP3α) in full medium was prepared at an equivalent MIP3α concentration of 1 µg/mL. Separately, a solution of free MIP3α at a matching concentration of 1 µg/mL, and a positive control solution of free MIP3α at 10 µg/mL, were prepared and distributed into the wells of a Transwell permeable 24-well plate (12x6.5 mm inserts; 8.0 µm PET membrane (Costar Corning, Kennebunk, USA). After 24 hours of incubation at 37 ºC, to allow sufficient MIP3α to be released from the NPs, the insert was pre-warmed with warm complete culture medium and the lower chamber solution was carefully re-suspended to homogenize MIP3α into the solution. Next, 1x105 RAW264.7 cells were carefully added to each upper chamber insert and allowed to migrate for 24 hours. Next, the cells were fixed with 4% PFA, washed and stained with a crystal violet solution, after which several digital pictures of each insert were acquired with a reverse microscope. Cell migration was quantified using Image J software v. 1.5. The migration index was calculated by dividing the area (%) of migrated cells by the area (%) of migrated cells induced by the positive control.
Pet membrane
Manufactured by Corning
Sourced in United States
The PET membrane is a laboratory equipment product designed for filtration applications. It is made of polyethylene terephthalate (PET) material and serves as a permeable barrier for the separation and purification of various substances.
Automatically generated - may contain errors
Lab products found in correlation
24 well cell culture insert, by Corning (2 mentions)
Alexa fluor 594 goat anti mouse igg, by Abcam (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Cell culture insert, by Corning (1 mentions)
Biocoat matrigel invasion chamber, by BD (1 mentions)
Fetal calf serum (fcs), by Merck Group (1 mentions)
Fetal calf serum (fcs), by Corning (1 mentions)
Histopaque 1077, by Merck Group (1 mentions)
Facscanto 2, by BD (1 mentions)
6 well companion plate, by Corning (1 mentions)
Axiocam microscope, by Zeiss (1 mentions)
Mirax slide scanner, by Zeiss (1 mentions)
Mircury rna isolation kit, by Qiagen (1 mentions)
Ultra low attachment six well plate, by Corning (1 mentions)
Rnase free dnase set, by Qiagen (1 mentions)
Inverted fluorescence microscope, by Olympus (1 mentions)
Biocoat matrigel invasion chamber, by Corning (1 mentions)
Diff quick, by Sysmex (1 mentions)
16 protocols using pet membrane
1
Measuring NP-Mediated Cell Migration
2
Cell Migration Assay with Plant Extract
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Briefly, CaSki cells were seeded into a 6-well plate, after they reached confluency, cells were serum starved for 24 h. Next day, cells were trypsinized and 1×10 5 of them resuspended in 250 µl of DMEM at 1% FBS were seeded in the upper chambers of the transwell insert (6.5 diameter, 8.0 µm pore size, PET membrane, Costar, Kennebunk, USA). Except for the control, all the cells were treated with different concentrations of the plant extract (15, 30, 40, 50 and 60 g/mL) chosen below its 24h IC 50 value. The lower chambers were filled with 750 µL DMEM with 10% FBS. The cells were allowed to migrate for 24h, afterward they were washed twice with PBS and fixed with 4% formaldehyde for 15 min. Following this, formaldehyde was removed, cells were washed twice with PBS and permeabilized at RT for 20 min using 100 % methanol. After permeabilization, the cells were again washed twice with PBS and stained with 0.2% crystal violet, incubated for 15 min at RT and washed with PBS. The non-migratory cells were removed from the top aspect of the membrane using cotton buds. For their quantification, cells were imaged under a Nikon Inverted Microscope ECLIPSE TE2000-S and counted using ImageJ software. All assays were performed at least three times.
3
CFTR Protein Detection in Lung Epithelial Cells
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CFBE41o− and 16HBE14o- were plated on a cell culture insert (0.75 × 106 cells per insert) containing a PET membrane (0.4 μm pore size) (www.corning.com ) to provide an air-liquid interface. Cells were transfected 12 h after plating with 5000 ng cmRNAhCFTR or equivalent (in nmol) pDNAhCFTR using Lipofectamine 2000 (www.invitrogen.com ) according to manufacturer’s instructions. Membranes were cut out from the insert 24 h after transfection, fixed with 4% PFA, blocked with 0.1% BSA and Fc blocker. Blocking was followed by overnight incubation with hCFTR clone 596 (1:250, kindly provided by the cystic fibrosis foundation therapeutics Inc.). As secondary antibody served Alexa Fluor 594 goat anti-mouse IgG (1:250, www.abcam.com , (ab150116)). Membranes were mounted on a coverslip and images were acquired by Zeiss Confocal Laser Scanning Microscope (CLSM) 710 NLO with Zen software.
4
Migration and Invasion Assays
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The migration and invasion assays were performed in 24-well cell culture inserts (Corning) fitted with a PET membrane (8 µm pore size). The inserts for invasion assays were coated with 30 µL of Matrigel matrix at 37°C for 1 h. Transfected cells were plated in medium without serum in the top chamber of a transwell. The bottom chamber contained 600 µL RPMI 1640 medium with 10% FBS. After incubating for 24 h at 37°C, the cells that had migrated to the lower surface of the membrane were fixed with 4% methanal, stained with crystal violet and photographed under a microscope. Cell numbers were counted under a light microscope at × 200 magnification.
5
Cancer Cell Invasion and Migration Assay
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The cancer cell invasion assay was performed in BD BioCoat™ Matrigel™ Invasion Chamber (8 μm pore size) (BD Bioscience, USA) and transwell migration assay was performed in cell culture inserts with PET membrane (Corning, USA). Assays were performed as instructed by the manufacturer’s protocols. Briefly, cells (1 × 105 cells) suspended in serum free medium were added to the upper chamber of an insert, and the insert was placed in a 24-well plate with DMEM supplemented with 10% fetal bovine serum. Migration assays were carried out for 6 h and invasion assays were carried out for 16 h. After the cells were incubated for exact time at 37°C, the inserts were washed with PBS, and cells on the upper surface of the insert were removed with a cotton swab. Cells adhering to the lower surface were fixed with 4% formaldehyde for 30 minutes, stained with 0.1% crystal violet solution and captured under a microscope.
6
Coculture of M1 and M2 Macrophages
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Lipopolysaccharide-induced M1Ms were plated at 1 × 106 cells per well in six-well plates and then cultured with 1 × 106 M2Ms, which were cocultured using six-well plates with 0.4-µm-pore, 10-μm-thick PET membrane (4.67 cm2 surface area) cell culture inserts (Transwell, Corning, NY, USA). A total of 2.6 mL of a suspension of 1 × 106 M1Ms was added to each plate well, and then 1.5 mL of a suspension containing 1 × 106 M2Ms was plated on the insert and incubated for 48 h under the same conditions (n = 3). Monoculture (M1Ms or M2Ms alone) was used as a control (n = 3). After incubation, medium and inserts were removed and M1Ms underwent total RNA extraction.
7
Cell Migration and Invasion Assay
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Migration and invasion assays were performed in 24‑well cell culture inserts (Corning) fitted with a PET membrane (8-μm pore size). The inserts for invasion assays were coated with 30 μL Matrigel matrix at 37 °C for 1 h. Transfected cells were plated in medium without serum in the top chamber of a transwell. The bottom chamber contained 600 μL RPMI 1640 medium with 10% FBS. After incubating for 24 h at 37 °C, the cells that had migrated to the lower surface of the membrane were fixed with 4% methanol, stained with crystal violet and photographed under a microscope. Cell numbers were counted under a light microscope at 200 × magnification.
8
Evaluating PDE4D's Impact on Pancreatic Cancer Migration and Invasion
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Cell migration and invasion assays were performed to test the effect of PDE4D on pancreatic cancer cells PANC1 and Bxpc3, using cell culture inserts with PET membrane (Corning, USA) and BD BioCoat™ Matrigel™ Invasion Chamber (8 µm pore size) (BD Bioscience , USA), respectively. 24 h after cells were transfected with PDE4D siRNAs (si-PDE4D-1 and si-PDE4D-2) or negative control siRNA (siNC), cell migration and invasion assays were performed according to the manufacturer's protocols and as previously described 20 (link). Migration assay and invasion assay were carried out at 7 h and 12 h for PANC1 cells respectively, while 20 h and 36 h for Bxpc3 cells respectively, after cells were suspended in serum free medium and added to the upper chambers of inserts.
9
Chemotaxis Assay of Monocyte Subsets
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Heparinized human peripheral blood was obtained from healthy donors (n = 3) after informed written consent approved by the cantonal ethics committee Zurich, Switzerland (KEK-ZH-2014-0430). Human peripheral blood mononuclear cells (hPBMCs) were isolated according to standard protocols by ficoll density gradient centrifugation (Histopaque-1077; Sigma, Switzerland). Migration of 3 × 106 hPBMCs in 300 μl basal medium supplemented with 0.5% fetal calf serum (FCS) (Sigma, Switzerland) towards the lower compartment containing either (I) 0.1 ml hMSC CM + 0.5% FCS (n = 5 per tissue source), (II) basal medium + 0.5% FCS or (III) 5 ng/ml monocyte chemoattractant protein 1 (MCP-1) + 0.5% FCS, was evaluated through a PET membrane with 3 μm pores (Corning, USA) after 3 h. The number of migrated cells was defined by counting cells over 1 min on a FACSCanto II (BD Bioscience, USA). Monocyte subpopulations were differentiated by staining with CD14, CD16, and propidium iodide (PI) (supplementary Table S2 ; supplementary Fig. S13 ).
10
Co-culture Lung Model Construction
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Co-culture lung models were comprised of 16HBE14o− cells and dTHP-1 macrophages. The models were constructed on 4.2 cm2 trans-well inserts with a PET membrane (3 μm pores) that were supported in a 6-well companion plate (Corning, Germany). Prior, to cell culture the apical side of the trans-well membranes were pre-treated with fibronectin solution. The first stage of the co-culture construction required the establishment of a stable 16HBE14o− epithelium on the apical side of the fibronectin coated trans-well by seeding at a concentration of 1 × 106 cells/ml for 7 days at 37 °C with 5% CO2. Subsequently, 500 μl of dTHP-1 cells (1 × 105 cells/ml) were placed on to the 16HBE14o− epithelial layer and allowed to adhere for 1.5 h. Following this step any excess macrophages in suspension were removed and replaced with 2 ml of fresh MEM culture medium. Co-culture models were allowed to further establish for 24 h prior to use (co-culture model structure is illustrated in Additional file 1 : Figure S1) [37 ].
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