Alexa fluor 488 conjugated egf

Cells were incubated for 48 h with 1,3,4-O-Bu₃ManNAc with serum starvation over the last 24 h. The cells then were washed with PBS, treated with enzyme free cell dissociation buffer (Life Technologies) until they detached from the culture plate, collected, and counted and cell numbers were normalized using the Beckman Z2 cell coulter counter. Cells were then washed twice in Live Cell Imaging Solution (Life Technologies) supplemented with 1.0% bovine serum albumin (BSA) and 20 mM glucose and treated with 0.5 μg/mL of filipin (Sigma Aldrich) or 100 mM of lactose (Carbosynth) for 60 min. Cells were then incubated at 37°C for 30 min with 2.0 μg/mL of Alexa Fluor 488-conjugated EGF (Life Technologies). Cells were washed three times, followed by acid washing for 5.0 min with 0.2 M glycine (pH 2.5), washed thrice and finally analyzed using flowcytometry with an Accuri C6 Flowcytometer. The cell population of interest was gated appropriately and 10⁴ cells falling within the gated area were measured and used to determine the mean fluorescence of the cell population; the histograms for these experiments are shown in Figure S6 in the Supplemental Material.

Cells were incubated for 48 h with 1,3,4-O-Bu₃ManNAc with serum starvation over the last 24 h. Cells were then washed in Live Cell Imaging Solution (Life Technologies) supplemented with 1.0% bovine serum albumin (BSA) and 20 mM glucose. Cells were then incubated at 37°C for 10 min or 30 min with 2.0 μg/mL of Alexa Fluor 488-conjugated EGF (Life Technologies). Cells were then fixed, nuclei were stained with DAPI and then imaged on Zeiss AxioObserver with 780-Quasar confocal module & FCS. Endosome size was estimated using ImageJ software based on published procedures (88,89).