An invasion assay was performed using 6.5-mm Transwell chambers with 8-μm pores (Costar) according to the manufacturer’s instructions. Briefly, 1 × 105 cells in serum-free medium were seeded in the upper insert precoated with matrigel (1:4, BD Biosciences), and then 600 μl complete medium containing 10 μg exosomes was added to the bottom chamber as a chemoattractant. After incubation for 24 h, the upper surface of each membrane was cleaned, and cells adhered to the insert surface were fixed and stained with 0.5% crystal violet.
6.5 mm transwell chambers with 8 μm pores
Manufactured by Corning
Sourced in United States
The 6.5-mm Transwell chambers with 8-μm pores are a type of lab equipment used for cell culture applications. They feature a permeable membrane with 8-micron pores that allow for the study of cell migration, invasion, and other cellular processes.
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10 protocols using 6.5 mm transwell chambers with 8 μm pores
1
Transwell Invasion Assay with Exosomes
2
Cell Cycle and Astrocyte Migration Assay
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The cell cycle assay was performed as described in our previous study [15 (link)]. Briefly, cells (5 × 105) were trypsinized, fixed in cold 70% ethanol for at least 1 h, and stored at -20°C. The fixed cells were then washed with PBS, treated with RNase (1 mg/mL), and stained with propidium iodide (50 mg/mL) for 30 min at 4°C, after which DNA content analysis was performed on an EPICS ELITE flow cytometer (Beckman Coulter, USA). The cell cycle distribution was analyzed using ModFit LT2.0 software. Astroctye migration was examined using 6.5 mm transwell chambers with 8-μm pores (Costar, Cambridge, MA, USA). A 100-μL medium containing dissociated astrocytes of 2 × 104 was then transferred to the top chambers of each transwell, and 600 μL of complete medium was added into the lower cell-free chambers. After allowing the cells to migrate for 16 h, non-migrated cells on the upper surface of each membrane were cleaned with a cotton swab. Cells adhering to the bottom surface of each membrane were stained with 0.1% crystal violet, imaged, and counted using a DMR inverted microscope (Leica Microsystems, Germany). Assays were performed three times using triplicate wells.
3
Transwell Migration Assay for Schwann Cells
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Cell migration was evaluated by Transwell migration assay. As described previously (Weinstein and Wu, 2001), 6.5-mm transwell chambers with 8-μm pores (Costar, Cambridge, MA, USA) were used to examine SC migration. The Transwell chamber membrane surface was pre-coated with 10 µg/mL fibronectin (Beyotime). SCs were resuspended in Dulbecco’s modified Eagle’s medium (1 × 106 cells/mL) and transferred onto the top of the Transwell chamber. After incubation at 37°C in 5% CO2 for 24 hours, SCs were then allowed to migrate to the lower chamber. The SCs were then stained with crystal violet (Beyotime) and counted using an inverted microscope (Leica Inverted Microsystems).
4
Transwell Assay for Cell Migration
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The migration ability of HCT116 cells was examined in 6.5 mm transwell chambers with 8 μm pores (Costar). Briefly, 600 μl DMEM supplemented with 10% FBS was added to the bottom chamber. 200 μl resuspended cells (2×106/ml) treated with compound in serum-free DMEM were added to the top chamber. After migration for 48 h, cleaned cells upper surface of the membrane with a cotton swab and washed with PBS. Then the chamber was stained with crystal violet (Bioworld) for 5 min. The quantity of cells on the bottom of the membrane was performed under a microscope (Leica Microsystems).
5
Transwell Migration Assay for Astrocytes
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Migration of astrocytes was studied using 6.5 mm transwell chambers with 8 μm pores (Corning Costar) as described previously (Dong et al., 2013 (link)). One hundred microliters astrocytes (2 × 105 cells/ml) resuspended in DMEM/F12 were transferred to the top chambers of each transwell and allowed to migrate at 30°C in 5% CO2 for 30 h, and 600 μl of DMEM/F12 was injected into the lower chambers. The upper surface of each membrane was cleaned with a cotton swab at the indicated time point. Cells adhering to the bottom surface of each membrane were stained with 0.1% crystal violet, imaged, and counted using a DMR inverted microscope (Leica Microsystems). Assays were done three times using triplicate wells.
6
Transwell Assay for Macrophage Migration
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Migration of RAW264.7 cells were measured using 6.5-mm transwell chambers with 8-μm pores (Costar, Cambridge, MA) as described previously [25 (link)]. A total of 100 μl of RAW264.7 cells (2 × 105 cells/ml) was transferred into the top chamber of the transwells and allowed to migrate at 37 °C in 5% CO2. Meanwhile, 600 μl of astrocytes (1 × 105 cells/ml) was seeded into the lower chambers. After migration for 48 h, the upper surface of each membrane was cleaned with a cotton swab. Cells attached to the bottom surface of each membrane were stained with 0.1% crystal violet, imaged, and counted using a DMR inverted microscope (Leica Microsystems, Bensheim, Germany). Assays were performed in triplicate for three times. To determine the effect of CCL5 on astrocyte-stimulated RAW264.7 migration, astrocytes were stimulated with 0.5 μg/ml recombinant MIF following CCL5 siRNA interference for 24 h prior to a transwell assay. For M2 macrophage transition, RAW264.7 cells were treated with or without 20 ng/ml rat recombinant IL-13 for 2 days.
7
Transwell Assay for SC Migration
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The migration ability of SCs was examined by a Transwell-based assay as described previously (14) . The 6.5-mm Transwell chambers with 8-μm pores (Costar, Cambridge, MA, USA) were used, and the bottom surface of each membrane was coated with 10 μg/ ml fibronectin. Primary SCs (10 6 cells/ml, 100 μl) were resuspended in DMEM, transferred to the top chamber of each Transwell, and allowed to migrate in a humidified 5% CO 2 incubator at 37°C with the complete medium (600 μl) being pipetted into the lower chamber. The upper surface of each membrane was cleaned with a cotton swab at indicated time points. The cells adhering to the bottom surface of each membrane were stained with 0.1% crystal violet (Beyotime, Shanghai, China), imaged, and counted under a DMR inverted microscope (Leica Microsystems). Assays were performed three times using triplicate wells.
8
Transwell Migration Assay for gAS
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The migration of gAS was studied using 6.5 mm transwell chambers with 8 μm pores (Corning Incorporated) as previously described (31 (link)). A total of 100 μl of gAS (2 × 105 cells/ml) with or without siRNA knockdown for 24 h was resuspended in DMEM followed by transfer to the top chambers of each transwell and allowed to migrate at 37 °C in 5% CO2. Moreover, 2.5 μg/ml recombinant gMIF was added into the lower chambers. After migration for 24 h, the upper surface of each membrane was cleaned with a cotton swab at the indicated time point. Cells adhering to the bottom surface of each membrane were stained with 0.1% crystal violet, imaged, and counted using a DMR inverted microscope (Leica Microsystems). Assays were performed three times using triplicate wells.
9
Cell Migration and Invasion Assay
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Cells were starved in serum-free DMEM (Thermo Fisher Scientific) for 24 h after transfection of 48 h, and then culturing was performed using 6.5 mm transwell chambers with 8 μm pores (Corning Incorporated, Corning, NY, USA). In brief, the bottom surface of each membrane was coated with fibronectin. Then, GC cell (1×105 cells) suspension was seeded into the upper chambers, and 600 μL of complete medium was added into the lower chambers. After incubating at 37°C for 24 h, the upper surface of each membrane was cleaned with a cotton swab. Cells adhering to the bottom surface of each membrane were fixed with 4% paraformaldehyde in phosphate-buffered saline and stained with 0.1% crystal violet. The migrated and invaded cells were counted and photographed at ×200 magnification in five random fields of view, and mean ± SD was calculated accordingly.
10
Astrocyte Migration Assay Protocol
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For the migration assay of astrocytes, the 6.5 mm transwell chambers with 8 μm pores (Corning Incorporated) were used as previously described [64 (link)]. Briefly, 100 μl of astrocytes (2 × 105 cells/ml) was resuspended in DMEM and transferred to the top chambers of each transwell. Meanwhile, 1 U/ml thrombin or 100 μM SFLLRN-NH2 was added into the lower chambers. The cells were allowed to migrate at 37 °C in 5% CO2 for 24 h, and the upper surface of each membrane was cleaned with a cotton swab. Cells adhering to the bottom surface of each membrane were stained with 0.1% crystal violet, imaged, and counted using a DMR inverted microscope (Leica Microsystems). Assays were performed three times using triplicate wells.
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