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TitleDEVELOPMENT OF QUANTUM DOT-BASED LIVE-CELL PHARMACOLOGICAL ASSAYS AIMED ...
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37


Labeling HEK Flp-In-293 Cells with Ligand-Conjugated Quantum Dots for Flow

Cytometry

The stably transfected hDAT-expressing HEK Flp-In-293 cells were treated

using a two-step QD labeling protocol (Figure 18). Prior flow cytometry experiments,

DAT-expressing HEK Flp-In-293 cells were seeded in 24-well polylysine-coated plates

(BD Bioscience®) and were allowed to grow for approximately 48 hours.

HEK Flp-In-293 cells were washed with warm KRH buffer and incubated with

GBR12909, high-affinity DAT antagonist, for 20 minutes at 37
o
C and 5% CO2. HEK

cells were then washed again with KRH buffer and incubated at room temperature for 10

minutes with equal parts IDT444 plus GBR12909 in desired concentration mixture or

only IDT444 of desired concentration. Following the ligand/drug incubation, the cells

were washed with KRH buffer. Once washed, HEK cells were incubated at RT for 5

minutes with 1nM of SavQD655 plus 1%BSA mixture (Qdot® 655 streptavidin

conjugate, Invitrogen). After the SavQD655 labeling step, HEK cells were washed with

KRH to remove any unbound SavQD655 and cells were nonenzymatically dissociated

from the 24-well polylysine-coated plates using CellStripper (Mediatech Inc.) and

transferred to polystyrene disposable tubes (BD Bioscience® Falcon tubes).

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