3 well removable chamber glass slides

The untransfected or transfected HEK 293 cell cultures were integrated into sandwich-type configurations. First, 3-well removable chamber glass slides (Ibidi) were coated with human fibronectin (Corning). Next, HEK 293 cells were seeded at densities of ~60,000 cells/cm² and were grown for ~14 to ~16 h. When necessary, the cells were transfected with vectors encoding for RfA1 for ~48 h (see Growth and transfection of human cells) or were exposed to the FBS-free growth media in the absence of any transfection reagents or vectors under the same conditions. Next, the untransfected or transfected cells were incubated for ~1 h in MEM supplemented with Earle’s salts, for which the NaCl concentration was adjusted to 117 or 217 mM. In turn, the substrates with monolayers at a ~50% to ~75% confluency were fixed with 3% PFA in PBS, thoroughly washed with PBS, treated with anti-fade mounting media (Ibidi), and covered (overlaid) with a thin glass coverslip. Note that the preparation and use of cell cultures within the ~50% to ~75% confluency window ensured rigorous quality control and facilitated comparisons across all of the experiments. The resulting configurations, which contained either fixed transfected or untransfected cells, were imaged with brightfield optical microscopy and characterized with reflectance and transmittance spectroscopy.

The untransfected or transfected HEK 293 cells were labeled with fluorescently-tagged wheat germ agglutinin according to standard protocols. First, the cells were seeded on 3-well removable chamber glass slides (Ibidi) coated with human fibronectin (Corning) at a density of ~60,000 cells/cm² and were grown for ~14 to ~16 h. When necessary, the HEK 293 cells were transfected with vectors encoding for RfA1 for ~48 h (see Growth and transfection of human cells) or were exposed to the FBS-free growth media in the absence of any transfection reagents or vectors under the same conditions. Next, the untransfected or transfected cells were incubated for ~1 h in MEM supplemented with Earle’s salts, for which the NaCl concentration was adjusted to 117 mM or 217 mM. In turn, the cells were stained with Alexa 555 fluorophore-conjugated wheat germ agglutinin (ThermoScientific) in Hank’s Balanced Salt Solution (HBSS) (ThermoScientific) and subsequently washed in pre-warmed HBSS. Finally, the cells were fixed with 3% PFA in 0.1 M PB. The resulting stained untransfected and transfected cells were imaged with fluorescence microscopy.