Visualizing Dose-Dependent Spike Protein Uptake with Quantum Dot Conjugates

תקציר

Source: Tran, B. N., et. al. High-throughput Confocal Imaging of Quantum Dot-Conjugated SARS-CoV-2 Spike Trimers to Track Binding and Endocytosis in HEK293T Cells. J. Vis. Exp. (2022)

This video demonstrates the use of quantum dot–conjugated spike protein (QD-Spike) to visualize dose-dependent viral entry in ACE2-GFP–expressing cells. Confocal imaging captures the internalization of QD-Spike via receptor-mediated endocytosis, modeling early coronavirus entry.

פרוטוקול

Treatment of cells with quantum dot–spike (QD-Spike)

1. Prepare 0.1% bovine serum albumin (BSA) by diluting in imaging media.
   1. For 10 mL of imaging media, add 130 µL of 7.5% BSA and mix.
2. In a 12-well reservoir or assay plate, dilute the 440 nM QD-Spike (SARS-CoV-2, Isolate USA-WA1/2020) stock to 20 nM using 0.1% BSA in imaging media.
   1. For 1 well of QD-Spike, add 2.27 µL of 440 nM QD-Spike to 47.73 µL of 0.1% BSA imaging media for a final concentration of 20 nM.
   2. To generate a six-point 1:3 serial dilution (in triplicate), add 14.26 µL of QD-Spike to 285.74 µL of 0.1% BSA imaging media to make the highest concentration of 20 nM. Add 100 µL of 20 nM QD-Spike to 200 µL of 0.1% BSA media to make the second dilution of 6.67 nM QD-Spike. Repeat four times to generate the six concentration points.
3. Using a multi-channel aspirator, remove all spent media from each well. Using a multi-channel pipette, wash once with imaging media (100 µL/well).
4. Aspirate 100 µL of imaging media and add back 50 µL/well of QD-Spike solution.
5. Incubate the plate for 3 h in a humidified incubator at 37 °C with 5% CO₂.

חומרים

רשימת החומרים שנעשה בהם שימוש במאמר זה

שם	חברה	מספר קטלוג	הערות
7.5% Bovine Serum Albumin	Gibco	15260-037	Used for stabilizing QDs in Optimem I and preventing non-specific interactions, final concentration 0.1%
Black clear bottom 96 well coated plate coated with poly-D-lysine	Greiner	655946	Microwell plates used for seeding cells and assaying QD-Spike
HEK293T hACE2-GFP	Codex Biosolutions	CB-97100-203	Human embryonic kidney cell line stably expression human angiotensin converting enzyme 2 tagged with GFP
Opti-MEM I Reduced Serum Medium	Gibco	11058-021	Imaging media, used for incubating cells with quantum dots
Quantum Dot 608 nm-Spike (QD608-Spike)	custom made by Naval Research Laboratory		Used for assaying SARS-Cov-2 Spike binding to hACE2 and monitoring Spike endocytosis