Considerations When Optimizing Coating Protocols for Corning ...

The micrographs below show that cell spreading wasimproved when SFM was used to dilute fibronectin ascompared to PBS (Fig. 2). The micrographs also show thatsimilar cell spreading can be achieved when cultured onfibronectin for 30 minutes or 24 hours, depending on theconcentration of fibronectin used.Laminin, another coating used to promote cell attachmentand spreading, was also tested under the same conditions asabove using Neuro-2a and NG108 cells (NG108 data notshown) (Fig. 3A). It was found that coating times of 24 hoursnegatively impacted cell spreading (data not shown). No differencewas observed in cell spreading regardless of diluentused. Various coating concentrations and coating times werealso compared using laminin that was coated at 37ºC and atroom temperature (25ºC) (Fig. 3B).DiscussionIn summary, we have found the effectiveness of biologicalcoatings to be directly related to coating concentration andcoating time. Our data suggests that similar results can beachieved with either a short coating time with high concentrationsor a long coating time with lower concentrations.By monitoring cell spreading of multiple cell lines dilutedwith various reagents, it was found that the effect on cellattachment can be dependent on the diluent used as well asthe temperature at which the coating was applied. Theseresults suggest that each diluent should be examined carefullywhen setting up experimental conditions. To improveexpermental results when coating Transwell permeable supports,we recommend optimizing coating time, concentrationand diluent used.Figure 2. HT1080 Cells on Fibronectin-Coated Transwell ® Permeable SupportsCoatingTimeCoatingConcentrationCoating CoatingPBS SFM Time Concentration PBS SFM10 µg/cm 210 µg/cm 224 hours2 µg/cm 230minutes2 µg/cm 20.4 µg/cm 20.4 µg/cm 210 µg/cm 2Uncoated6 hours2 µg/cm 20.4 µg/cm 2