To test the effect of L48H37 on the migratory potential and invasive ability of U2OS and MG-63 cells in vitro, we employed a modified Boyden chamber migration assay with and without Matrigel coating, respectively. After treatment with L48H37 with concentrations of 0, 1.25, 2.5, and 5 μM, the cells were seeded into the upper section of the Boyden chamber (Neuro Probe, Cabin John, MD, USA) at a density of 2 × 105/mL for U2OS cells and 4 × 105/mL for MG-63 cells, and then the U2OS cells and MG-63 cells were incubated in the modified Boyden chamber migration assay and invasion assay at 37 °C for 24 h and 48 h, respectively. Under a light microscope, the migratory and invasive cells were finally counted, as stated previously [32 (link),37 (link),38 (link),39 (link)].
Boyden chamber
Manufactured by Neuro Probe
Sourced in United States
The Boyden chamber is a device used to study cell migration and invasion. It consists of two compartments separated by a porous membrane. Cells are placed in the upper compartment, and a chemoattractant or other stimulant is placed in the lower compartment. The cells migrate through the membrane in response to the stimulus, and their movement can be quantified.
Automatically generated - may contain errors
Lab products found in correlation
Matrigel, by BD (16 mentions)
Polycarbonate membrane, by Neuro Probe (5 mentions)
Trypsin, by Thermo Fisher Scientific (4 mentions)
Human fibronectin, by Merck Group (4 mentions)
Giemsa, by Merck Group (3 mentions)
Hema 3 staining kit, by Thermo Fisher Scientific (3 mentions)
Giemsa solution, by Merck Group (3 mentions)
Gimmsa, by Merck Group (2 mentions)
Ix71 microscope, by Olympus (2 mentions)
Boyden chambers, by R&D Systems (2 mentions)
Murine recombinant chemokine c c motif ligand 3 ccl3, by R&D Systems (2 mentions)
Eclipse e200 microscopy, by Nikon (2 mentions)
Giemsa s solution, by Merck Group (2 mentions)
Ckx41, by Olympus (1 mentions)
Percoll gradient, by Merck Group (1 mentions)
Transwell insert, by Merck Group (1 mentions)
Bacto agar, by BD (1 mentions)
Eclipse ts2, by Nikon (1 mentions)
Ckx41 microscope, by Olympus (1 mentions)
Pd98059, by Merck Group (1 mentions)
79 protocols using boyden chamber
1
Evaluating L48H37 Impact on Cell Migration
2
Exosome-Induced Migration Assay for A549 Cells
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To evaluate the functional effect on lung epithelial cells (A549) induced by HMC-1 derived exosomes, reverse migration assay was performed using a 46 well Boyden chamber (Neuroprobe Inc.). Briefly, A549 cells (32,500 cells/well) were added in the lower chamber and allowed to adhere onto the gelatin (0.1%) coated polycarbonate membrane by inverting the Boyden chamber upside down (Neuroprobe Inc.). After 3 hours of incubation the assembly was placed in correct orientation and exposed to various HMC-1 exosomes dosage on the upper side of the membrane for 12 hours. Migrating cells towards the upper side of membrane were fixed in ethanol, and stained with Giemsa (Histolab, Gothenburg, Sweden) and images were acquired using a light microscope (Zeiss Axioplan, Germany). Adhered cells, present on lower side of membrane (non-migrated side), were wiped out carefully before imaging.
3
Thymoquinone Inhibits Cell Invasion and Migration
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786-O-SI3 cells were treated with Thymoquinone at the indicated concentrations for 24 h. Otherwise, cells were pretreated with Thymoquinone (0-20 μM) for 2 h followed by incubated with or without 10 ng/mL TGF-β1 for an additional 48 h. After treatment, the cells were harvested and seeded in a Boyden chamber (Neuro Probe, Cabin John, MD, USA) at a cell density of 104 cells/well and then incubated in serum-free medium at 37°C for 12 h. For the invasion assessment, 10 μL Matrigel® (BD Biosciences, Bedford, MA, USA) was applied into the membrane filters (pore size 8 μm, Neuro Probe, Cabin John, MD, USA) and the standard medium was added into the bottom chamber of the apparatus. After 24 h incubation, the filters were dried in a laminar flow hood, then the invaded cells were fixed with methanol and stained with Giemsa. Cell numbers were counted using a light microscope (CKX41; Olympus). The transmigration assessment was performed as described in the invasion assay except for use of Matrigel 20 (link).
4
Neutrophil Chemotaxis Assay for Adipose Tissue
5
Boyden Chamber Migration Assay
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The migration assay of SCC-9 cells was performed using Boyden chamber (Neuro Probe, Cabin John, MD, USA) for 24 h as described by Chung et al [47 (link)]. SCC-14 cells were seeded on Transwell inserts (Millipore, Bedford, MA, USA) at 104 cells/well in serum-free medium and placed in the upper chamber then incubated for 48 h. For invasion assay, 10 μl Matrigel (25 mg/50 ml; BD Biosciences, MA, USA) was applied to polycarbonate membrane filters and the bottom chamber contained standard medium. The cell migratory abilities were determined by counting the migrated cells in five fields under high magnification.
6
VEGF-A-Induced Cell Migration Assay
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Cell migration toward VEGF-A was analyzed using a Boyden chamber (Neuro Probe, Gaithersburg, MD, USA), as previously described.(27 (link)) VEGF-A (10 ng/mL) was added to the lower chamber as a chemoattractant. TEC were treated with the control siRNA (10 nM) or siSBSN (10 nM) in endothelial basal medium (EBM)-2 supplemented with 0.5% FBS for 24 h. In total, 1.5 × 104cells were seeded in the upper chamber and incubated for 4 h at 37°C. The assays were independently performed three times.
7
Quantifying Cell Migration and Invasion
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The cell migration assay was performed according to the methods described by Neoh et al. [56 (link)]. HA22T cells were seeded into a Boyden chamber (Neuro Probe, Cabin John, MD, USA) at 104 cells/well in serum-free media. HA22T cells with 11-epi-SA treatment (0, 2.66, 5.32 and 7.98 μM) were kept at 37 °C for 24 h to allow cell migration. For invasion assay, 10 μL Matrigel (25 mg/50 mL; BD Biosciences, MA, USA) was coated onto 8 μm pore-size polycarbonate membrane filters, and HA22T cells were plated in the upper chamber of the Matrigel-coated Transwell insert. The bottom chamber contained cell culture medium previously described by Yeh et al. [57 (link)]. The migrated and invaded cells on the lower chamber were fixed with 100% methanol and stained with 5% Giemsa (Merck, Germany). Cell numbers were counted using a 100× light microscope.
8
LLC Cell Soft Agar Assay and Migration Evaluation
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LLC cells (2 × 104) in
500 μl of D-MEM (20% FBS) were mixed to
500 μl of 0.33% Bacto Agar (Difco Laboratories, Detroit,
MI) containing increasing concentrations of DHS. Each mixture was poured in
culture cell dishes previously prepared with 5 mL of 0.6% Bacto Agar in complete
D-MEM and incubated at 37 °C for 2 weeks. Five hundred
μl of D-MEM with 60% FBS were added twice a week to each cell dish.
The colonies formed were stained with 500 μl of 0.005%
Gentian Violet for 1 h, counted using a 10X magnitude inverted microscope (Leitz
DM-IL, Leica). To determine LLC cell migration or invasion, the Boyden chamber
(Neuroprobe, Gaithersburg, MD) was assembled by inserting collagen -
(100 μg/mL) or Matrigel
(200 μg/mL, BD Biosciences)-coated filters,
respectively, as previously described13 (link).
500 μl of D-MEM (20% FBS) were mixed to
500 μl of 0.33% Bacto Agar (Difco Laboratories, Detroit,
MI) containing increasing concentrations of DHS. Each mixture was poured in
culture cell dishes previously prepared with 5 mL of 0.6% Bacto Agar in complete
D-MEM and incubated at 37 °C for 2 weeks. Five hundred
μl of D-MEM with 60% FBS were added twice a week to each cell dish.
The colonies formed were stained with 500 μl of 0.005%
Gentian Violet for 1 h, counted using a 10X magnitude inverted microscope (Leitz
DM-IL, Leica). To determine LLC cell migration or invasion, the Boyden chamber
(Neuroprobe, Gaithersburg, MD) was assembled by inserting collagen -
(100 μg/mL) or Matrigel
(200 μg/mL, BD Biosciences)-coated filters,
respectively, as previously described13 (link).
9
Cell Migration and Invasion Assay
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Cell migration was assessed using a technique as previously described in the method by [16 (link)]. We seeded 5 × 104 cells, treated with the drug in serum-free media, into a Boyden chamber (Neuro Probe; Cabin John, MD, USA). These were then incubated at 37°C for 24 hours, allowing migrating cells to move through the membrane.
To evaluate cell invasion, we employed a method involving Transwell inserts with 8 μm pore-size polycarbonate membrane filters, precoated with 0.5 mg/mL Matrigel, as previously described [16 (link)]. Cells suspended in serum-free media were placed in the upper chamber, while the lower chamber contained serum-enriched media. Following the incubation period, membranes from the bottom chamber were washed with PBS and treated with 1 mg/ml MTT solution and then incubated at 37°C for an hour. The resultant formazan crystals were dissolved in DMSO, and absorbance was measured at 550 nm.
To evaluate cell invasion, we employed a method involving Transwell inserts with 8 μm pore-size polycarbonate membrane filters, precoated with 0.5 mg/mL Matrigel, as previously described [16 (link)]. Cells suspended in serum-free media were placed in the upper chamber, while the lower chamber contained serum-enriched media. Following the incubation period, membranes from the bottom chamber were washed with PBS and treated with 1 mg/ml MTT solution and then incubated at 37°C for an hour. The resultant formazan crystals were dissolved in DMSO, and absorbance was measured at 550 nm.
10
Transwell Invasion Assay for PC3 Cells
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An invasion test was performed as previously described using a 48-well Boyden chamber containing 8 μm polycarbonate filters (Neuroprobe, Inc., Gaithersburg, MD, USA). An invasion assay was performed in invasion chambers containing a membrane coated with Matrigel™. Untreated or UniPR1331-treated PC3 cells were added (75,000 cells/50 μL) to the top of each chamber and allowed to invade through coated filters for 6 h. In the lower compartment of the invasion chamber, 7% FBS-containing medium was added as a chemo-attractant. At the end of the incubation, the migrated/invaded cells attached to the lower membrane surface and were fixed, stained with Diff Quick (MBT), and counted with standard optical microscopy. The results of three separate experiments are presented as the mean ± SD.
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