For migration assay, the cells were incubated for 48 h post‐transfection and then, seeded on the membrane surface of the upper chamber (24‐well, Corning, Corning, NY, USA) coated without Matrigel (BD Biosciences), whereas the lower chamber (Corning) was added with DMEM with 10% FBS to undertake the nutritional attractant. For the invasion assay, the whole process was similar to migration assay except that the membrane of upper chamber was added with Matrigel. After 48 h, the cells which had migrated or invaded into the bottom surface of the membrane were dyed with crystal violet (0.5% w/v) for 30 min after the fixation with methanol. The invaded or migrated cells were counted using a Countess automatic cell counter (Invitrogen) in five randomly chosen fields under inverted microscope (Nikon TE‐300).
Lower chamber
Manufactured by Corning
Sourced in United States
The lower chamber is a component of Corning's laboratory equipment. It serves as the primary enclosure for conducting experiments or housing samples. The lower chamber provides a controlled environment to facilitate various research and testing applications.
Automatically generated - may contain errors
Lab products found in correlation
Upper chamber, by Corning (9 mentions)
Matrigel, by Corning (4 mentions)
Matrigel, by BD (4 mentions)
Axio observer z1, by Zeiss (3 mentions)
Inverted fluorescence microscope, by Olympus (2 mentions)
Crystal violet staining solution, by Beyotime (2 mentions)
Inverted microscope, by Nikon (2 mentions)
Fetal bovine serum (fbs), by Corning (2 mentions)
Countess automatic cell counter, by Thermo Fisher Scientific (1 mentions)
Te300, by Nikon (1 mentions)
Upper chamber, by BD (1 mentions)
Optical microscope, by Olympus (1 mentions)
High glucose dmem, by Corning (1 mentions)
Upper transwell chamber, by Corning (1 mentions)
Inverted microscope, by Olympus (1 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
Imatinib, by MedChemExpress (1 mentions)
Top chamber, by Corning (1 mentions)
Transwell chamber, by Corning (1 mentions)
Fibronectin, by Corning (1 mentions)
23 protocols using lower chamber
1
Evaluating Cell Migration and Invasion
2
Cell Migration and Invasion Assays
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Migration and invasion assays for cells with PIN1KD or ATRA treatments were carried out with the Boyden Chamber system; 200 μL of 5 × 104 cells starved in media with 1% FBS were placed in the upper chamber and 500 mL of media with 10% PBS were placed in the lower chamber (Corning). The cells were allowed to migrate for 24 h or 48 h and stained with crystal violet. For invasion assays, cells were inserted into the upper chamber with a Matrigel coated membrane (Sigma, St.Louis, MO, USA). Migrated cells adhering to the underside of the inserts were photographed (AXIO Observer Z1, Carl Zeiss, Gottingen, Germany) and counted.
3
Cell Invasion Assay Protocol
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For invasion, the transfected cells were seeded into an upper chamber (8 μm), cultured with 200 µl serum-free medium. Complete medium (600 µl) supplemented with 10% FBS was added into lower chamber (Corning, Inc.). The cells were incubated at 37°C for 24 h, followed by the fixation and staining of Crystal Violet Staining Solution (Beyotime, Shanghai, China) at RT for 30 min. Finally, cells were observed under an inverted fluorescence microscope (magnification, ×200; Olympus Corporation).
4
Cell Migration Assay Protocol
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In the migration test, the transfected cells (1×105) were inoculated into the top chamber (8 microns) with 200 µl serum-free medium. Complete medium (600 µl) containing 10% FBS was added to the lower chamber (Corning, Inc.). Following incubation at 37°C for 24 h, the migratory cells located under the insert were fixed and stained with Crystal Violet Staining Solution (Beyotime Institute of Biotechnology) at room temperature for 30 min and observed using an inverted fluorescence microscope (magnification, ×200; Olympus Corporation).
5
Transwell Assays for Migration and Invasion
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Transwell assays were used to evaluate the cell migration and invasion abilities. For the migration assay, 1 × 105 cells were suspended in 500 μl serum-free RPIM-1640 medium and seeded into the upper chamber (Corning, Corning, NY, USA), and 700 μl RPIM-1640 containing 10% Fetal Bovine Serum(FBS) was added to the lower chamber. After 24 h incubation at 37°C, the cells on the lower surface were fixed with paraformaldehyde, stained with 0.2% crystal violet, and then imaged and enumerated with an inverted microscope (Nikon, Tokyo, Japan). For invasion assay, the upper chamber was coated with Matrigel (BD, Franklin Lakes, NJ, USA) before cells seeded, and the other steps were the same as for migration assay.
6
Cell Migration and Invasion Assays
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For cell migration assays, 500 μL of conditioned medium from siRNA-treated cells was added to the lower chamber (Corning, USA), and 3×105 human umbilical vein endothelial cells (HUVECs) without FBS were seeded into the upper chamber. For cell invasion assays, Matrigel was diluted with RPMI-1640 and evenly spread on the bottom of the upper chamber. After the Matrigel solidified, 3×105 HUVECs in 100 μl of RPMI-1640 basal medium were added to the upper chamber, while 600 μL of conditioned medium from the siRNA cells was added to the lower chamber. After 24 hours and 48 hours of incubation for the migration and invasion assays, the transwell inserts were fixed with 4% paraformaldehyde. Finally, the inserts were dyed with 0.1% crystal violet solution for 20 min, and the invading cells were observed under an optical microscope (Olympus, Japan).
7
Wound Healing and Cell Migration Assays
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Cells plated in 6-well plates at 90% confluence were wounded with sterile 200-μL pipette tips. After wounding, the cells were washed twice with PBS to remove cell debris and incubated in serum-free medium. The cell-free wound area was photographed every 24 h at 50 × magnification. The motility speed of cells was determined using Image J software (National Institutes of Health, Bethesda, MD, United States) as an average closed area of the wound relative to the initial wound area at 48 h after wounding.
The Transwell assay was carried out to evaluate cell migration and invasion. Cells were resuspended in serum-free medium, and seeded in the upper chamber with an 8-μm pore size filter membrane at 2 × 105 cells/mL (100 μL/chamber), while conditioned medium with 200 mL/L fetal bovine serum was added to the lower chamber (Corning, Inc., Corning, NY, United States). The cell invasion assay was similar to the cell migration assay, except that upper chambers were coated with Matrigel (Corning, Inc.). The cells were incubated for 48 h in a 37 °C atmosphere containing 50 mL/L CO2. Cells in the upper chamber were removed with cotton swabs, whereas migrated/invaded cells on the bottom side of the membrane were fixed in 40 mL/L paraformaldehyde, stained with GIEMSA, and counted in five randomly selected microscopic fields (100 ×) per well. All experiments were carried out in triplicate.
The Transwell assay was carried out to evaluate cell migration and invasion. Cells were resuspended in serum-free medium, and seeded in the upper chamber with an 8-μm pore size filter membrane at 2 × 105 cells/mL (100 μL/chamber), while conditioned medium with 200 mL/L fetal bovine serum was added to the lower chamber (Corning, Inc., Corning, NY, United States). The cell invasion assay was similar to the cell migration assay, except that upper chambers were coated with Matrigel (Corning, Inc.). The cells were incubated for 48 h in a 37 °C atmosphere containing 50 mL/L CO2. Cells in the upper chamber were removed with cotton swabs, whereas migrated/invaded cells on the bottom side of the membrane were fixed in 40 mL/L paraformaldehyde, stained with GIEMSA, and counted in five randomly selected microscopic fields (100 ×) per well. All experiments were carried out in triplicate.
8
Transwell Invasion Assay for TNBC
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After dilution by serum-free and high-glucose DMEM (Biological Industries, USA) at the ratio of 1:8, 100 μl Matrigel (Corning, USA) was paved onto the center of upper transwell chamber (Corning, USA). Then 1×105 TNBC cells were supplemented onto the coagulated Matrigel, and 700 μl 10% FBS-inclusive high-glucose DMEM was poured into the lower chamber (Corning, USA). Twenty-four hours later, MDA-MB-231 and MDA-MB-468 cell lines were managed by 10% methanol for 20 min, followed by dyeing with 0.5% crystal violet for 30 min. Eventually, TNBC cells that hardly penetrated the upper chamber were wiped off, and 5 fields were randomly selected to count cell number under inverted microscope (Nikon, Japan).
9
Transwell Assay for Analyzing Cell Migration and Invasion
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Transwell assay was performed as mentioned previously to analyze cell migration and invasion ability [43 (link)]. Transfected cells were seeded into Transwell upper-chamber with serum-free medium, while medium containing 10% FBS was placed into lower chamber (Corning). For Transwell invasion assay, the chamber should be precoated with a thin-layer Matrigel (BD Biosciences) before cells were seeded. Consequently, the upper cells of chambers were removed using a cotton buds, and the bottom surface cells of chambers were fixed, stained and photographed.
10
Cell Migration Assay Protocol
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The transfected cells in serum‐free medium and the medium with 10% FBS were placed in the upper chamber (Corning, NewYork, NY, USA) and the lower chamber, respectively. After 24 hours of incubation, the cells that passed through the membrane were fixed and stained with methanol and 0.1% crystal violet and then photographed and counted using an inverted microscope (Olympus, Tokyo, Japan). The experiment included three replicates.
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