• 6 Well Glass Bottom Plate with high performance #1.5 cover glass (e.g., In Vitro Scientific, cat. no. P06-1.5H-N, http://www.cellvis.com/6-well-glass-bottom-plate-with-high-performance-number-1.5-cover-glass/product_detail.php?product_id=55)
CRITICAL: Note that most high resolution microscope objective lenses are corrected for #1.5 coverslips (unless they are equipped with an adjustable correction collar). Imaging with other thickness coverslips (i.e., #1) will induce spherical image aberrations that degrade image quality.
• Dow Corning 184 SYLGARD 0.5 kg 1.1 lb kit (Krayden, cat. no. DC4019862, http://krayden.com/buy/dc-184-sylgard-0-5kg-1-1lb-kit.html)
• 0.5 g of 3.11 µm non-functionalized polystyrene beads (Bangs Laboratories, cat. no. PS05N, http://www.bangslabs.com/products/ordering/list/PS05N)
CRITICAL: Bangs Laboratories is well-known for their quality control on their bead tolerances. Beads purchased from any other company should be checked for their consistency.
• 0.5 g of 5.15 µm non-functionalized polystyrene beads (Bangs Laboratories, cat. no. PS06N, http://www.bangslabs.com/products/ordering/list/PS06N)
• Human melanoma A375 cells (American Type Culture Collection, cat. no. CRL-1619, http://www.atcc.org/products/all/CRL-1619.aspx)
CRITICAL: Any other cell line of interest is acceptable. We use A375 cells because of our interest in melanoma metastasis.
o Appropriate culture media for A375 cells consists of
DMEM (Dulbecco’s Modified Eagle Medium), high glucose, pyruvate, without L-glutamine (Life Technologies, cat. no.10313-021,
GlutaMAX Supplement (Life Technologies, cat. no. 35050-061,
FBS (Fetal Bovine Serum), qualified, One Shot format, US origin (Life Technologies, cat. no. 26140-111,
Antibiotic-Antimycotic (Life Technologies, cat. no. 15240-096,
• Tissue culture hood and tissue culture incubator capable of maintaining 37oC, 100% humidity and 95% air/5% CO2 conditions.
• HEPES 1M (Life Technologies, cat. no. 15630-080,
o Required for buffering media for live-cell imaging.
• Bovine Serum Albumin (Sigma-Aldrich, cat. no. A9418,
o For coating PDMS and coverslip surfaces.
• Nalgene Rapid-Flow Sterile Disposable Filter Units with PES Membrane (Thermo Scientific, cat. no. 566-0020, http://www.thermoscientific.com/en/product/nalgene-rapid-flow-sterile-disposable-filter-units-pes-membrane.html)
• Vacuum system
• Vacuum Desiccator
• Pipettes and tips (including serological pipettes)
• Rhodamine B isothiocyanate dextran MW 70 kDa (Sigma-Aldrich, cat. no. R9379,
o For validating the thickness of confinement chambers as described above.