For cell invasion assay, trans-well filters (Costar) were coated with matrigel (3.9 μg/μl, 60–80 μl) on the upper surface of a polycarbonic membrane (6.5 mm diameter, 8 μm pore size). After 24 h post-transfection and then 6 h serum starvation, A549 and Calu-3 cells (2 × 105) were resuspended in 200 μl of serum-free medium in the upper chambers, and while 500 μl media containing 10 % FBS (as a chemoattractant) was added to the bottom chamber. After incubating for 8 h at 37 °C in a humidified incubator with 5 % CO2, cells in the upper chamber were carefully removed with a cotton swab. Cells that had migrated to the basal side of the membrane were fixed with methanol, stained with hematoxylin, mounted and dried at 80 °C for 30 min. We randomly selected three visual fields and counted and recorded the number of cells invading the matrigel with an inverted microscope at 200× magnification. Each test was performed in triplicate. With the similar principle and approach, we used trans-well filters (Costar) with uncoated matrigel on the upper surface of a polycarbonic membrane (6.5 mm diameter, 8 μm pore size) for cell migration assay.
Transwell filter
Manufactured by Corning
Sourced in United States, Germany, United Kingdom, France
Transwell filters are a type of laboratory equipment used for cell culture and migration studies. They consist of a membrane insert that separates two chambers, allowing the movement of cells, molecules, or other substances between the chambers to be observed and measured.
Automatically generated - may contain errors
Lab products found in correlation
Matrigel, by BD (32 mentions)
Matrigel, by Corning (12 mentions)
Dmem f12, by Thermo Fisher Scientific (7 mentions)
Fitc dextran, by Merck Group (7 mentions)
Crystal violet, by Beyotime (6 mentions)
Inverted microscope, by Olympus (6 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (5 mentions)
Fibronectin, by Merck Group (5 mentions)
Glutamax, by Thermo Fisher Scientific (4 mentions)
Matrigel basement membrane matrix, by BD (4 mentions)
Crystal violet, by Merck Group (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Crystal violet, by Solarbio (3 mentions)
Penicillin streptomycin, by Merck Group (3 mentions)
Inverted microscope, by Zeiss (2 mentions)
Microplate reader, by Molecular Devices (2 mentions)
Ccl19, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
307 protocols using transwell filter
1
Cell Invasion and Migration Assay
2
Exosome Permeability and Migration Assays
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For the in vitro permeability assay, 3×104 HUVECs were added to upper chambers of 24-well Transwell filters (Corning, 0.4 µm, cat. NO. 3470) and treated with culture medium or exosomes from SCLC cells for 48 hours with HUVECs reaching 100% confluence. Fluorescein isothiocyanate-dextran (FITC-dextran, Sigma, cat. NO FD70S) was then added to upper chambers at 1 mg/mL. Medium in the lower chamber was collected at 30 min, 60 and 90 min successively, to detect its fluorescence intensity with 490 nm excitation and 520 nm emission.
For the transendothelial migration assay, 3×104 HUVECs were seeded on upper chambers of 24-well Transwell filters (Corning, 8 µm, cat. NO. 3422) and treated with SCLC-cell-derived exosomes for 48 hours. After the vascular endothelial cells reached 100% confluence, GFP+ H446 cells were added in upper chambers (2×104 cells/well). Culture medium containing 20% FBS was added to lower chambers for transendothelial migration of GFP+ H446 cells. After twelve hours of migration, cells remaining on upper chambers were swabbed, cells migrated through filters were counted under a fluorescence microscope.
For the transendothelial migration assay, 3×104 HUVECs were seeded on upper chambers of 24-well Transwell filters (Corning, 8 µm, cat. NO. 3422) and treated with SCLC-cell-derived exosomes for 48 hours. After the vascular endothelial cells reached 100% confluence, GFP+ H446 cells were added in upper chambers (2×104 cells/well). Culture medium containing 20% FBS was added to lower chambers for transendothelial migration of GFP+ H446 cells. After twelve hours of migration, cells remaining on upper chambers were swabbed, cells migrated through filters were counted under a fluorescence microscope.
3
Transwell Assay for Cell Invasion
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell invasion was evaluated with 8-µm Transwell filters (Corning Inc., Corning, NY, USA). The upper chambers of the Transwell filters were coated with Matrigel (Corning Inc.) and incubated at 37°C for at least 30 min. Then, 4×104 cells were seeded on the Matrigel-coated filter with RPMI-1640 plus 1% FBS, and RPMI-1640 containing 10% FBS was added to the lower chamber. After 24 or 36 h, non-migrating cells were carefully removed from the upper chamber with a cotton swab, and the filters were fixed and stained with 0.01% crystal violet solution for 1 min. Invading cells were photographed, and the invaded cell proportion was determined by measuring the OD570 nm value of the crystal violet eluted with ethanol.
4
Transwell Migration and Invasion Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To perform the migration assay, 5 × 104 cells per well were seeded into the upper chambers of Transwell filter (Corning Costar, Acton, MA, USA) with serum-free RPMI 1640 medium. To conduct the invasion assay, 1 × 105 cells were seeded in the upper chambers of the filters coated with 1 mg/mL Matrigel (BD Biosciences, San Jose, CA, USA). Then, 600 µL of RPMI-1640 medium containing 20% FBS was added to the lower chamber. After 12, 24 and 48 h of incubation, to examine migration and invasion, the non-migrated cells on the upper surface of the membrane were removed, and the invading cells below the membrane were fixed with 4% paraformaldehyde for 20 min and stained with hematoxylin-eosin (HE) at 37 °C for 15 min. Under a 100× inverted microscope, five fields on each membrane were randomly selected, and the mean number of penetrating cells was calculated.
5
Transwell Cell Migration Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell migration was measured according to the ability of the cells to migrate across a transwell filter (8 μm pores, Costar, Cambridge, MA, USA). 1 × 105 cells suspended in serum-free medium were added to the upper chamber and medium containing 10 % FBS was added to the lower chamber. After incubation in 37°C for 24 h, the non-migrated cells were scraped off the filter using a cotton swab. The cells that migrated to the lower side from the upper chamber were fixed with 4 % paraformaldehyde and stained with hematoxylin. The cells per microscopic field were taken pictures (20×) and counted in 8 randomly chosen fields.
6
Transwell Cell Migration Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Transwell cell migration assay was performed as described previously [14 (link), 16 (link)]. HEK293T cells were transfected with YFP alone, or together with the indicated plasmids and incubated for 24 h. The cells were detached and then resuspended in serum free DMEM. The cells were replated onto the upper chamber of a Transwell filter (Costar, 8 μm pore size). DMEM supplemented with 10% FBS was added to the lower chamber. At 6 h after plating, cells were fixed with 4% paraformaldehyde in phosphate buffered saline. Non-migrated cells on the upper side of the filter were removed with a cotton swab. In parallel, cells were separately plated to culture wells without the Transwell filters for estimating the total number of attached cells. Cell migration was calculated by the number of YFP-positive cells underside of the filter normalized to the total number of the YFP-positive attached cells. For each experiment, the number of cells in at least 8 random fields on the underside of the filter was counted, and four or five independent filters were analyzed.
7
Breast Cancer Cell Migration Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
EpRAS mouse breast cancer cells were pretreated for 10 d with TGF-β1 and/or asporin peptide (P159–205), as described above. At the end of this period, 1 × 105 cells were suspended in serum-free medium (0.1% BSA, 1% penicillin/streptomycin) and seeded into the upper part of a Transwell filter (diameter 6.5 mm, pore size 8 μm; Costar, catalog no. 3422). The lower compartment was filled with DMEM containing 1% pen/strep and 10% FBS. Following 16 h of incubation at 37°C, migrating cells were fixed and stained with Diff-Quick kit (Reagena, catalog no. 102164). Pictures of each insert were taken at 5× magnification, and migrating cells were counted using ImageJ software (US National Institutes of Health).
8
Cell Migration Assay with Transwells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HeLa cells were released from plates and then resuspended in serum-free α- minimum essential media (α-MEM(–)). The cells (2 × 104 cells) were replated onto the upper chamber of a Transwell filter (Costar; 8-μm pore size). The cultured medium containing serum was added to the lower chamber. After 6 h of incubation at 37 °C, transwells were removed from the plates and cells in the bottom wells were stained with calcein-AM (Dojindo). The fluorescence intensity in each well was determined using a Wallac 1420 Victor2 Microplate Reader (PerkinElmer). No peptide represents the fluorescence intensity of the wells for the cell similarly incubated in α-MEM(–) without containing peptides.
9
Macrophage Polarization Regulation by MSCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Monocytes were isolated from the bone marrow, and erythrocytes were removed with Red Blood Cell Lysis Buffer (Beyotime, China) for 5 min and then washed with RPMI 1640 + 10% FBS three times. The cells were filtered with a 70-μm strainer and cultured for 2 h in RPMI 1640 + 10% FBS before being switched into RPMI 1640 + 10% FBS with 25 ng/ml macrophage colony-stimulating factor (M-CSF, AF-400-28, Peprotech) for 4 days. Then, the differentiated BMDMs were co-cultured with MSCs infected with Vector or IL-33 lentivirus or MSCs only at a 10:1 ratio with a transwell filter (3-μm pore size; Costar, USA). After 48 h of co-culture, the macrophages were collected for flow cytometry analysis of macrophage polarization with macrophage common marker anti-rat CD68 (Bio-Rad, USA), anti-rat iNOS (Abcam, USA) for M1, and anti-rat CD206 (Proteintech, USA) for M2. Flow cytometry assays were conducted using a Millipore Guava® easyCyte 8 (Millipore, USA), and data were analyzed using Guava InCyte™ software.
10
Transwell Cell Migration Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells migration assays were measured using transwell filter with 8.0 μm pores (Costar, USA). Cells at a density of 50,000 cells were seeded in the upper chamber including 200 ul serum-free DMEM/F12 medium, while 600 ul complete medium was added into the lower chamber. After culturing for 2 days, the cells were fixed with 4% paraformaldehyde and stained via 5% crystal violet solution. The migrated cells were photographed with a light microscope and analyzed using ImageJ software.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!