These clumps were then transferred to an ultra-low attachment non-tissue culture treated plate (Corning, cat# 3471) and cultured in suspension.
For the TGF-β R3 ELISA assay, capture antibody (R&D systems, cat: AF-242-PB) was immobilized onto an EIA/RIA plate (Corning, cat: 3590) overnight.
In experiments using cultured polarized EC, cells were grown on 12-mm collagen-coated Transwell inserts with 0.4 μm pore PTFE membranes (Corning, Cat. no. 3493).
Approximately 2 × 105 cells/ml (in 80 ml of Sf-900III medium) were added to a 250 ml Erlenmeyer flask (Corning, Cat. No. 431046) and placed on a shaker platform at 100 rpm.
The probe was incubated in solution at 95°C for 5 minutes, then lightly centrifuged for 2 minutes to collect condensation, and cooled to room temperature for 20 minutes, and placed in a hybridization chamber (Corning, cat. No.2551).
A 24-well plate of 8.0 μm pore-size diameter polycarbonate (PC) membrane transwell inserts (Corning, Cat. # 3422, USA) were pretreated with complete DMEM/F12 culture medium prior to cell inoculation.
After co-culture, the cells were transferred to fluorescence-activated cell sorter tubes (Corning, Cat. No. 352235), washed with 0.5% BSA-PBS, and centrifuged at 300 × g for 5 min.
They were printed at a final concentration of 250 ng/μl on amino-silane coated slides (Corning Cat # 40005) in quadruplicate at room temperature and 50% relative humidity using a PixSys 5500 microarrayer (Genomic Solutions) fitted with Majer Precision Pins.
For immunofluorescent analysis of SVEC4-10 cells, the cells were grown on 12-mm Transwells with 0.4 μm Pore Polyester Membrane Insert (Corning, Cat. no. 3460), as described in Section 2.3, until they reached confluence, and then were washed with PBS and fixed in 3.7% paraformaldehyde for 10 minutes at room temperature.
1×105/well HBMEC in 200 µL medium were seeded onto polycarbonate membrane inserts (pore size 0.4 µm, surface area 0.33 cm2) purchased from Corning Inc. (Cat. no. 3413, Corning, NY).
