Costar tissue culture plates

Murine BaF3 lymphocytes expressing murine FGFR1IIIc were a gift from Professor David Ornitz (Washington University, St. Louis, USA). Cells were maintained in RPMI-1640 supplemented + 10% FCS + 100 Units/ml penicillin G + 100 μg/ml streptomycin sulfate (all from ThermoFisher, UK) and 1 ng/ml murine interleukin-3 (R&D Systems, UK) at 10⁵ to 10⁶ cells/ml at 37 °C, 5% CO₂. For assays, cells were plated at 10⁵ cells/ml in 96-well, flat bottomed Costar tissue culture plates (Corning, USA) in 100 μl growth medium without interleukin-3 supplemented with FGF-2 (R&D Systems, UK) and heparan sulfate at the indicated concentrations. Cells were incubated for 72 h at 37 °C, 5% CO₂. Thiazolyl blue tetrazolium bromide (Sigma, UK) was then added to a final concentration of 250 μg/ml and the cells were incubated a further 4 h at 37 °C, 5% CO₂. The assay was stopped with the addition of 50 μl 10% SDS, 0.01 N HCl and the plates were incubated 4 h to overnight at 37 °C to dissolve the formazan product. Plates were read at 570 nm using a Thermo multiskan EX plate reader (ThermoFisher, UK).

The AV canal explants were cultured on collagen gels for analysis of EMT essentially as previously described (Xiong et al., 2012 ). Briefly, collagen gels were made from a solution of type I rat tail collagen (Corning, Corning, NY, USA) in 12-well Costar tissue culture plates (Corning). Collagen gels were soaked with culture medium containing OptiMEM-1 (Thermo Fisher Scientific, Waltham, MA, USA), 1% FBS (Thermo Fisher Scientific), 100 U/ml penicillin (Thermo Fisher Scientific) and 100 µg/ml streptomycin (Thermo Fisher Scientific) overnight at 37°C. The culture medium was removed from the gels prior to the dissection of the AV canals. AV canals were dissected from E9.5 embryos, transferred to the collagen gels, and incubated for 48 h in 5% CO₂ at 37°C. At 24 h and 48 h of culture, images were acquired using an Axiovert 40 CFL inverted microscope equipped with an AxioCam digital camera and imaging system (Carl Zeiss Microscopy). Elongated or spindle-shaped mesenchymal cells located outside of the endocardial sheath were counted as migrating mesenchymal cells. For statistical analysis, unpaired two-tailed Student's t-test was used (error bars in associated graphs show the s.d.).

Leukocytes were resuspended at 3.5×10⁶ cells/ml in HL-1 media (1% L-Glutamine, 1% Penicillin Streptomycin, 2.5% β-Mercaptoethanol) and 100 µl/well was plated out in triplicate on 96 well Costar tissue culture plates (Corning Incorporated, USA). The cells were stimulated with 100 µl/well of, appropriate antigen, positive controls of 5 µg/ml Con A (Sigma-Aldrich, USA) or 25 ng/ml of SEB (Sigma-Aldrich, USA) or negative controls of medium alone. The plates were incubated at 37°C, 5% CO₂ for 5 days. Eight hours prior to harvesting, 1 µCi/well of [³H]-Thymidine (GE Healthcare, UK) was added. The cells were harvested onto fiberglass filtermats (PerkinElmer, USA) using a Harvester 96 plate harvester (Tomtec, USA) and counted on a Wallac Betaplate scintillation counter (EG&G Instruments, Netherlands). Results were expressed as stimulation index (SI) (cpm of stimulated cells divided by cpm of negative control cells). An SI of ≥2.5 was considered to indicate a positive proliferation response.