PC-3, PC/DX25, DU-145, and DU/DX50 cells were examined for their invasion ability using an invasion assay with Growth Factor Reduced BD BioCoat Matrigel invasion chambers (BD Biosciences, San Jose, CA, USA) according to the manufacturer’s instructions. PC-3, PC/DX25, DU-145, and DU/DX50 cells were placed in the upper chamber and seeded at a density of 1 × 104 in serum free RPMI medium (500 μL). Complete medium (1000 μL) was placed in the lower chamber. The cells were incubated at 37 °C and 5% CO2 for 12 to 16 h. Cells were then fixed in iced methanol for five to 10 min and stained with hematoxylin for 15 to 30 min. Cotton-tipped swabs were used to remove the cells on the upper side of the filters, and the filters were washed with dH2O. A microscope was used to examine and count cells on the underside of the filters. Each condition was plated in triplicate for each experiment. All experiments were repeated for at least three times.
Growth factor reduced biocoat matrigel invasion chambers
Manufactured by BD
Sourced in United States
The Growth Factor Reduced BD BioCoat Matrigel invasion chambers are a laboratory equipment designed to measure the invasive potential of cells. These chambers provide a standardized in vitro system to assess cell invasion through a reconstituted basement membrane matrix.
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Lab products found in correlation
Control mouse igg, by R&D Systems (1 mentions)
Il 6 blocking antibody, by R&D Systems (1 mentions)
Control goat igg, by R&D Systems (1 mentions)
Slgv004sl, by Merck Group (1 mentions)
Diff quik cell staining kit, by Sysmex (1 mentions)
Diff quik stain kit, by Sysmex (1 mentions)
Transwell kit, by BD (1 mentions)
4 protocols using growth factor reduced biocoat matrigel invasion chambers
1
Invasion Assay for Prostate Cancer Cell Lines
2
In vitro invasion assay with FABP4
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An in vitro invasion assay was performed in triplicate using Growth Factor Reduced BD BioCoat Matrigel Invasion Chambers (BD Biosciences) according to the manufacturer’s instructions. Briefly, 3 × 104 cells were seeded in the upper chamber with conditioned medium from PrSC treated with or without 100ng ml-1 rFABP4, and the presence or absence of 10 μg ml-1 of IL-8 blocking antibody, IL-6 blocking antibody, control goat IgG, or control mouse IgG (R&D Systems, Minneapolis, MN, USA). In the siRNA experiments, cells were treated with 50 nM FABP4 siRNAs for 24 hours before being seeded in the chambers. Subsequently, 20% FBS DMEM was placed in the lower chamber, followed by incubation for 24 hours. In some experiments, 1 × 104 PrSC were seeded in the lower chamber with optimal medium. Sera from mice were collected, clarified by filtration (SLGV004SL; Millipore, Billerica, MA, USA), and used for ex vivo cell invasion assays. Briefly, 3 × 104 cells were seeded in the upper chamber with medium containing 5% FBS or 5% mouse serum with or without 10 μg ml-1 of IL-8 blocking antibody or 30 μM of BMS309403. DMEM with 20% FBS was placed in the lower chamber. Then, the non-invading cells in the upper chamber were removed and the membranes were stained with a Diff-Quik cell-staining kit (Sysmex, Kobe, Japan) to count the invading cells. The experiments were performed twice each in triplicate.
3
In Vitro Matrigel Invasion Assay
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The in vitro invasion assay was carried out in Growth Factor Reduced BD BioCoat™ Matrigel™ invasion chambers (BD Biosciences) according to the manufacturer’s instructions. Briefly, cells (1 × 104) were seeded in serum-free medium in the upper compartment, and Dulbecco’s Modified Eagle’s Medium supplemented with 20% FBS was used a chemoattractant in the lower compartment of the chamber. After 22 h, non-invasive cells on the upper side of the chamber were removed, and the membranes were stained using a Diff-Quik Stain Kit (Sysmex Corp., Hyogo, Japan). Invasive capacity was quantitatively analyzed by measuring the number of invasive cells under a light microscope. Each assay was performed in triplicate.
4
Transwell Migration and Invasion Assays for Prostate Cancer Cells
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Migration assays for control, AR siRNA knockdown, or Casodex‐treated (5 μmol/L Casodex treatment for three passages) C4‐2B cells were carried out following the instructions of the Transwell kit purchased from BD Bioscience (Franklin Lakes, NJ, USA) as previously described.31 Cells were seeded at a concentration of 2 × 104 in 500 μL serum‐free RPMI medium into the upper compartment of Transwell. The lower compartment of Transwell was filled with 500 μL RPMI medium containing 10% FBS. After 18 hours incubation, cells that migrated to the lower surface of the filter were fixed with 100% methanol and stained with 1% Giemsa solution. Total migrated cells were counted following standard procedures. Invasion assay of control or AR siRNA knockdown C4‐2B cells was carried out with Growth Factor Reduced BD BioCoat Matrigel invasion chambers according to the manufacturer's instructions (BD Bioscience) as previously described.31 Cells were seeded at a concentration of 2 × 104 in 500 μL serum‐free RPMI medium into the upper compartment of Transwell. The lower compartment of Transwell was filled with 500 μL RPMI medium containing 10% FBS. After 18 hours incubation, cells that migrated to the lower surface of the filter were fixed with 100% methanol and stained with 1% Giemsa solution. Total migrated cells were counted following standard procedures.
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