A cell migration assay was carried out to measure the chemotactic activity of SDF-1α using Costar polycarbonate Transwell inserts (pore size 5 µm, diameter 6.5 mm, Costar # 3421). SDF-1α was diluted in 0.6 mL of migration medium [RPMI-1640 medium containing 0.5% BSA (Sigma # A9576)] and added to wells in a 24-well plate (“bottom wells”). Transwell inserts (“top wells”) were placed into the bottom wells and loaded with 5×105 Jurkat cells suspended in 100 µL migration medium. After 2 h incubation at 37°C, cells that transmigrated into the bottom well were mixed and counted with a BD Accuri C6 flow cytometer (BD Biosciences). Each sample was counted twice at a preset flow rate (14 µL/min for 10 µL). For negative controls, the cells were added to the top wells with no SDF-1α in the bottom wells. For the input cell number, cells were added directly to the bottom well and counted. Migration was calculated as a percentage of the input cell number after subtraction of the numbers in negative controls.
Polycarbonate transwell inserts
Manufactured by Corning
Sourced in United States
Polycarbonate transwell inserts are a type of lab equipment used to study cell migration, invasion, and permeability in vitro. They consist of a porous polycarbonate membrane that allows for the separation of two cell culture compartments, enabling the observation of specific cellular behaviors across the membrane.
Automatically generated - may contain errors
Lab products found in correlation
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Accuri c6 flow cytometer, by BD (1 mentions)
Collagen type 1, by Merck Group (1 mentions)
Facscanto 2, by BD (1 mentions)
Cxcl12, by BioLegend (1 mentions)
Crystal violet, by Beyotime (1 mentions)
Dmem f12 media, by Thermo Fisher Scientific (1 mentions)
Matrigel, by Corning (1 mentions)
Celltiter glo, by Promega (1 mentions)
Trypsin neutralizing solution, by Lonza (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by GE Healthcare (1 mentions)
24 well transwell chamber, by Corning (1 mentions)
Matrigel, by BD (1 mentions)
Elx 800 type, by Agilent Technologies (1 mentions)
Celltracker green cmfda, by Thermo Fisher Scientific (1 mentions)
24 well plate, by Corning (1 mentions)
Prolong gold mounting medium, by Thermo Fisher Scientific (1 mentions)
21 protocols using polycarbonate transwell inserts
1
Transwell Chemotaxis Assay for SDF-1α
2
Transient PI3K-C2β Downregulation Impacts Cell Migration
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Cells were seeded in a 6 well plate and serum starved overnight. To determine the effect of transient downregulation of PI3K-C2β, cells were transfected with the specific siRNA and serum starved overnight the day following the transfection. Where indicated polycarbonate Transwell inserts (8.0 μm diameter pores, 6.5 mm diameter, Costar®) were coated overnight with 100 μg/ml Type I collagen (Sigma Aldrich) in PBS at +4 °C. Coated or uncoated inserts were then incubated in 500 μl RPMI supplemented with 0.5% BSA for 1 hour at 37 °C/5% CO2 atmosphere. Cells were resuspended in RPMI supplemented with 0.5% BSA and plated on inserts (4,000 cells for collagen-induced migration; 12,000 or 20,000 cells for FBS-induced migration). The lower chamber was filled with 500 μl of media supplemented with 10% FBS (uncoated inserts) or serum free media (coated inserts) and cells were left to migrate for 24 h (FBS) or 4 h (collagen) at 37 °C/5% CO2 atmosphere. Non-migrated cells were removed from the upper chamber using a cotton bud while cells that had migrated were fixed using 4% paraformaldehyde for 30 min and stained with 0.1% crystal violet solution for 15 minutes. Pictures were taken from different fields using a light microscope at 10× magnification and cells were counted using the Image J program. A minimum of 10 fields were counted. All experiments were performed in duplicate.
3
Transwell Assay for CLL Cell Migration
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Transmigration of the CLL cells was assessed using polycarbonate Transwell inserts with a 5 μm pore size (Corning Costar). Briefly, the s-CLL and l-CLL cells sorted at 1 × 106/mL each were placed in the upper chamber in the RPMI-1640 medium containing 1% bovine serum albumin (BSA). Inserts were placed into the lower chamber containing RPMI-1640 with 1% BSA in the presence or absence of 200 ng/mL of CXCL12 (BioLegend). After 3 h at 37 °C in 5% CO2, the cells migrated into the lower chamber and were counted using a BD FACSCanto II instrument. The migration rate was calculated as the ratio of CXCL12-treated to CXCL12-untreated cells that transmigrated through the insert.
4
Chemotaxis Assay of MSCs Migration
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Chemotaxis experiments were performed in polycarbonate transwell inserts (8-µm pores; Corning Costar Corp., Corning, NY, USA). RLE-6TN cells (1×106) were added in the lower chamber. MSCs (1×105) in 100 µL of culture medium were seeded into the upper chamber in serum-free medium and cultured at 37°C for 24 h. The non-migrating cells in the upper chamber were carefully removed with a cotton swab. Migrating cells were fixed in 4% paraformaldehyde, stained with 1% crystal violet (Beyotime Institute of Biotechnology, Shanghai, China), and photographed. The number of migrated cells was calculated as the ratio of the experimental samples to the control samples ×100, by counting at least 5 randomly separated fields.
5
MDCK Cell Polarization in Transwell and Coverslip
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MDCK cells polarized in Transwell inserts: MDCK cells (100,000–400,000 cells) were plated on 12-mm or 24-mm polycarbonate Transwell inserts with 0.4 mm pore size (Costar) and grown with daily media changes until transepithelial resistance reached 400 ohm x cm2 (4–5 days), as measured using an EVOM electrometer (World Precision Instruments, Sarasota, FL). MDCK cells polarized in glass coverslips: MDCK cells (5000 cells) were plated in 12-mm-diameter glass coverslips and used for the experiments 3–4 days after achieving full confluence. Under both conditions, the cells developed primary cilia, Rab11-ARE punctate compartment subapically localized, and distribution of basolateral markers, as characteristics of the polarized phenotype (Casanova et al., 1999 (link); Brown et al., 2000 (link); Folsch et al., 2009 (link); Perez Bay et al., 2013 (link)).
6
Quantifying Bacterial Adherence to Enteroids
7
Transfection of hMSC Sheets with Lipoplexes
8
Quantifying Cell Migration in Transwell Assays
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Equal numbers of cells (50,000) were seeded onto polycarbonate Transwell inserts (Corning, NY) with transparent, permeable (8 μm pore) filters. Cells were placed in 4 mL of Ham's F‐12 media supplemented with 0.5% FBS and 1% penicillin/streptomycin. Cells were allowed to adhere for 1–2 h before addition of drugs. After 24 h of drug (or vehicle) treatment, filters were washed with PBS, and cells fixed in ice cold 95% ethanol for 10 min. Following fixation, cells were washed with PBS and stained with brilliant blue (Sigma) for 5 min at room temperature. Filters were washed and placed in PBS. Each filter was examined via a microscope‐mounted camera under 4× magnification and images of at least five fields per filter were obtained at random using Q‐capture software to measure total number of cells. The top of the filters were then scraped with a cotton swab, rinsed with PBS to remove nonadherent cells, and images of at least five fields containing migrated cells were again obtained at random. Once images of all fields were obtained, the number of cells in each image was counted. Cell migration was expressed as a percent of total cells.
9
Motility and Invasion Assays with miRNA
10
Co-culture Transwell Assay for Cell Interaction
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The human fibroblast line CCC-ESF-1 (ESF) was obtained from the Cell Center of the Chinese Academy of Medical Sciences (Beijing, China). The human breast cancer cell line BT474 was acquired from the Cell Resource Center of the Chinese Academy of Sciences. ESF or BT474 cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Invitrogen; Thermo Fisher Scientific, Waltham, MA, USA) with 10% fetal bovine serum (GE Healthcare, Buckinghamshire, UK), 10 µg/ml streptomycin and 100 U/ml penicillin (both purchased form Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C in a humidified 5% CO2 atmosphere. ESF and BT474 cells were co-cultured using polycarbonate Transwell inserts (0.4 µm pore size; Corning Inc., Corning, NY, USA). ESF cells (2×105 cells/well) were plated at the bottom of 6 well companion culture plates and then allowed to adhere for at least 2 h without apical Transwell inserts. ESF cells plated in the wells were exposed to BT474 cell-conditioned media by placing the Transwell inserts plated with BT474 cells (1.5×105 cells/well) into these wells. This method allowed the ESF and BT474 cells to grow in the same medium without direct contact between them. Each experiment was repeated 3 times.
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