4.5. Flow Cytometry Analysis of Cellular Uptake

WT K562 and SH-SY5Y cells, SDC transfectants and differentiated SH-SY5Y cells were utilized to quantify the internalization of fluorescently labeled Aβ1–42 or ApoEs. Briefly, 6 × 10⁵ cells/mL in DMEM/F12 medium (with 10% FCS) were incubated with FITC-labeled Aβ1–42 or ApoEs (at a concentration of 5 μM and 500 nM, respectively) for various amounts of time (3 and 18 h) at 37 °C. The effect of ApoEs on Aβ1–42 uptake was measured by preincubating the cells with either of the unlabeled ApoE isoforms for 30 min at 37 °C at a concentration of 500 nM, before treating the cells with 5 μM of FITC-labeled Aβ1–42. After incubation, the cells were washed twice in ice-cold PBS and progressed towards flow cytometry. Then the cells (WT K562, SH-SY5Y and SDC transfectants) were resuspended in 0.5 mL of physiological saline. Equal volumes of this suspension and a stock solution of trypan blue (Merck KGaA, Darmstadt, Germany; 500 μg/mL dissolved in ice-cold 0.1 M citrate buffer at pH 4.0) were allowed to mix for 1 min before the flow cytometric analyses. In this way, the sample’s pH was lowered to pH 4.0, thereby optimizing the quenching effect of trypan blue to quench the extracellular fluorescence of surface-bound fluorescent proteins [14,16]. Cellular uptake and attachment was then measured by flow cytometry using an FACScan (Becton Dickinson, Franklin Lakes, New Jersey, USA). Cellular attachment was calculated by subtracting intracellular fluorescence (i.e., those quenched with trypan blue or trypsin) from the measures of overall fluorescence. A minimum of 10,000 events per sample was analyzed. The viability of cells was determined by appropriate gating in a forward scatter against side scatter plot to exclude dead cells and debris [14,16].