To explore whether the expression of AEG-1 affected cell migration and invasion, Transwell migration and invasion assays were performed [40 (link), 47 (link)]. For the migration assay, exponentially growing cells (5 × 105 cells/mL) were seeded into the upper chamber (BD Bioscience) in culture medium with 1% FBS, whereas for the invasion assay, the upper chamber was pre-coated with Matrigel (BD Bioscience) prior to adding the cells (5 × 105 cells/mL), and then they were all incubated for 24 h at 37°C. The lower chamber was added with 600 μL RPMI 1640 containing 10% FBS. Then, the cells on the upper surface of the filters would be scraped off with swabs. After fixed the membrane with 4% paraformaldehyde and stained with crystal violet, the cells were taken photos under microscope. At least 5 microscopic fields were counted for each transwell filter.
Upper chamber
Manufactured by BD
Sourced in United States
The Upper Chamber is a specialized laboratory equipment component designed to facilitate specific experimental procedures. It serves a core function within the overall laboratory setup, but a detailed description of its intended use would require further information to maintain an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Matrigel, by BD (39 mentions)
Upper chamber, by Corning (9 mentions)
Matrigel, by Corning (7 mentions)
Matrigel, by Merck Group (7 mentions)
Inverted microscope, by Olympus (7 mentions)
Hematoxylin solution, by Merck Group (5 mentions)
Crystal violet, by Merck Group (5 mentions)
Inverted light microscope, by Olympus (4 mentions)
Upper chamber, by Merck Group (4 mentions)
Matrigel pre coated, by Merck Group (3 mentions)
Cell counting kit 8 (cck8), by Dojindo Laboratories (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Matrigel, by Solarbio (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
8 μm pore size chamber inserts, by Corning (1 mentions)
Crystal violet, by Beyotime (1 mentions)
Magnificationx100, by Olympus (1 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
Fetal calf serum (fcs), by Merck Group (1 mentions)
Lower chamber, by Corning (1 mentions)
104 protocols using upper chamber
1
Cell Migration and Invasion Assays
2
Matrigel-Based Cell Invasion Assay
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Before inoculation of cells, 50 μL diluted Matrigel (BD Biosciences, Franklin Lakes, NJ, USA) was covered in polycarbonate membrane of the upper chamber and placed at 37° C (30 min) until gel was formed. Cells (5 × 104) were seeded in the upper chamber (BD Biosciences) containing serum-free medium (100 μL). Then, the basolateral chamber was added with 500 μL serum containing 20% FBS, and developed with 5% CO2 (37° C; 48 h). Gel and cells on the upper chamber were wiped out using a cotton swab after culture. Invasive cells were fixed with methanol, stained with 0.1% crystal violet and finally photographed and counted using microscope (× 100).
3
Transwell Assay for Cell Invasion and Migration
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Cell invasion and migration were evaluated using Transwell assays. the upper chamber (BD Biosciences) were precoated with or without Matrigel (cat. no. M8370; Beijing Solarbio Science and Technology Co., Ltd.) for 30 mins at 37˚C. Chambers that were coated with Matrigel were used for invasion assays whilst those without were used for migration assays. Briefly, the transfected RCC cells with serum-free medium were seeded into the upper chamber (BD Biosciences) at a density of 1.0x105 cells/ml. The lower chamber was added with medium supplemented with 10% FBS. Cells that invaded or migrated through the membrane were subsequently stained with 500 µl 0.1% crystal violet at 37˚C for 30 min after incubation for 36 h. The cells were observed using a light microscope (magnification, x100). Five different fields were observed and photographed. The relative cell migration and invasion rates were counted through the number of the migrated or invaded cells/the number of the inoculated cells in the same field.
4
Transwell Assay for Cancer Cell Migration and Invasion
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The capability of migration and invasion in indicated cancer cells were evaluated by transwell assays. Briefly, for invasion assay, 5 × 10^4 cells were suspended in serum-free medium and seed into the upper chamber (8-μm pore size, BD Biosciences, San Jose, CA, USA)) with diluted Matrigel (Corning, NY, USA). Medium supplemented plus 20% fetal bovine serum was added to the lower chambers. After incubation for 48 h, wiped off cells remained in the upper chamber. Then fixed cells with 4% paraformaldehyde followed by stained with 0.1% crystal violet. The cells migrated or invaded to lower chamber were counted and imaged in three different fields with a microscope.
5
Transwell Assay for Cell Migration and Invasion
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Cell migration and invasion were analyzed using Transwell chamber assays. For the migration assay, infected cells (1 × 105 cells per well) were suspended in serum-free medium and plated on the upper chamber (BD Biosciences, Eugene, OR, USA). For the invasion assay, infected cells were seeded into the upper chamber that was precoated with Matrigel. For both assays, the lower chamber was filled with DMEM containing 10% FBS. After 24 h of incubation, cells adhering to the lower membrane were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet solution for 15 min. Cells were enumerated by counting six random fields per Transwell chamber under a light microscope (magnification: 100×).
6
Boyden Chamber Migration and Invasion Assays
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Migration and invasion assays were performed using the Boyden chamber technique [35 (link)]. For the migration assay, approximately 1.5 × 105 of cells in 100 μL of serum-free medium were placed in the upper chamber (Corning Costar, NY, USA), which was not coated with Matrigel®, whereas 500 μL of the same medium with 10% FBS was placed in the lower chamber. After 24 h, the cells that had migrated were fixed with methanol, stained with crystal violet solution, and counted under a microscope using five random fields (magnification: 100×). For the invasion assay, a procedure described in the cell migration assay was performed, except that the upper chamber was pre-coated with Matrigel® (BD Bioscience, CA, USA). Data were analyzed using the student's t-test and P < 0.05 was considered statistically significant.
7
Cell Migration and Invasion Assay
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After being starved for 24 hours, 2 × 105 cells with the serum‐free medium were seeded into the upper chamber (8‐μm pore size, BD Bioscience, USA) pre‐coated with or without Matrigel (BD Bioscience, USA). Complete medium containing 10% FBS was added to the bottom of each well as a chemoattractant. After being cultured for 24 hours, non‐migrated or non‐invaded cells were removed off the upper chamber using a cotton swab while the migratory or invasive cells were counted after fixation with 4% paraformaldehyde and being stained with 0.1% crystal violet.
8
Cell Migration and Invasion Assay
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The transwell migration and invasion assay was performed as previously described [3 (link), 4 (link)] with transfected cells seeded into the upper chamber (BD Biosciences, MA) with or without a Matrigel coating and DMEM/F12 with 10% FBS in the lower compartment acting as a chemoattractant. Cells were allowed to migrate for 12 and 24 hours in the migration and invasion assays, respectively. The non-motile cells were removed from the top, and the cells in the bottom chamber were stained and counted under a light microscope. Relative migration and invasion activities are expressed as the fold-change over their respective controls. The effect of IQGAP1 or miR-124 on proliferation was measured using Cell Counting Kit-8 (Dojindo, Japan). Briefly, 5 × 103 cells were plated in 96-well plates for 24 hours and then transfected with the IQGAP1 cDNA vector/IQGAP1 siRNA, miR-124 mimics/inhibitors, and their respective controls. At 72 hours, the absorbance of the cells was measured with a spectrophotometer at 450 nm.
9
Transwell-based Cell Migration and Invasion Assays
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Transwell assays were utilized to assess the in vitro migration and invasion ability of cells.
To assess migratory ability with transwell assays, cells were transferred for 24 h and digested, followed by resuspension of MNK45 and SGC‐7901 cells using serum‐free RPMI‐1640. 1 × 105 cells were seeded to the upper chamber (BD Biosciences), and RPMI‐1640 medium containing 10% fetal bovine serum was added to the bottom chamber. 48 h later, migrant cells were fixed with 4% paraformaldehyde solution and stained with 10% crystal violet solution at room temperature. Five random visual fields were selected for photographing (100×) with a light microscope, and the Images software was used for quantitation.
To assess invasion ability with transwell assays, MNK45 and SGC‐7901 cells were digested with trypsin, collected, and resuspended in serum‐free medium, with concentration adjusted to 1 × 106 cells/ml. After being diluted at 1:5 using RPMI 1640 medium, Matrigel (Corning) was used to coat the upper chamber of transwell and allowed to dry at room temperature. The following steps are the same as above. Eventually, the number of cells stained with crystal violet under microscope was the invasion cell count.
To assess migratory ability with transwell assays, cells were transferred for 24 h and digested, followed by resuspension of MNK45 and SGC‐7901 cells using serum‐free RPMI‐1640. 1 × 105 cells were seeded to the upper chamber (BD Biosciences), and RPMI‐1640 medium containing 10% fetal bovine serum was added to the bottom chamber. 48 h later, migrant cells were fixed with 4% paraformaldehyde solution and stained with 10% crystal violet solution at room temperature. Five random visual fields were selected for photographing (100×) with a light microscope, and the Images software was used for quantitation.
To assess invasion ability with transwell assays, MNK45 and SGC‐7901 cells were digested with trypsin, collected, and resuspended in serum‐free medium, with concentration adjusted to 1 × 106 cells/ml. After being diluted at 1:5 using RPMI 1640 medium, Matrigel (Corning) was used to coat the upper chamber of transwell and allowed to dry at room temperature. The following steps are the same as above. Eventually, the number of cells stained with crystal violet under microscope was the invasion cell count.
10
Cell Migration Assay Protocol
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In total, 1 × 105 cells were seeded into the upper chamber (BD Biosciences, San Jose, CA, USA) with a serum-free medium. Medium (10% FBS, Invitrogen) was added to the lower chamber, and the medium served as a chemoattractant. After incubation at 37°C and 5% CO2 for 48 h, the chambers were placed in 4% paraformaldehyde for 30 min and then stained with 2% crystal violet for 30 min. The numbers of migrated cells were calculated in five random fields per well.
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