JoVE Encyclopedia of Experiments
Microbiology
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Begin with tubes containing human lung epithelial cells.
Add biotinylated influenza viruses and incubate on ice. The viruses bind to the cell surface without being internalized.
Centrifuge to separate the virus-bound cells, remove the supernatant and resuspend.
In the initial time point tube, add unlabeled streptavidin, which binds to the biotinylated viruses, forming complexes.
Add fluorophore-labeled streptavidin, which fails to bind to the streptavidin-biotin virus complex. Wash and fix the cells.
Incubate the later time-point tube at a higher temperature, enabling virus internalization, then return to ice.
Add unlabeled streptavidin and an internalization-blocking agent, which prevents further viral uptake and incubate. Streptavidin binds surface-bound biotinylated viruses, then wash.
Fix the cells and add a detergent-containing buffer to permeabilize them.
Wash and add fluorophore-labeled streptavidin, which enters the cells and binds to biotin.
Analyze the samples by flow cytometry.
A higher fluorescence in the later time point confirms successful viral internalization.
After repeating the wash one more time, cool the tubes down on ice for 10 minutes. When the tubes have cooled, spin again and use 100 microliters of biotinylated virus diluted in infection PBS to resuspend the pellets after incubating on ice for one hour, repeat the spin and wash with PBS twice, processing the following samples in parallel as much as possible for the zero minute samples.
To treat the zero minute plus STV samples, spin the tubes, remove the supernatant and use 15 microliters of STV 2% BSA and 0.1% sodium azide in PBS to resuspend the cell pellet. Incubate on ice for 30 minutes. Then centrifuge and use PBS to wash the cells twice after fixation with PFA as just demonstrated, store the samples at four degrees Celsius.
For the 30 minutes plus STV samples. After pelleting, resuspend the samples in 200 microliters of PBS 2% BSA, following an incubation at 37 degrees Celsius for 30 minutes.
Spin and resuspend the cells in 50 microliters of STV 2% BSA 0.1% Sodium azide in PBS incubate on ice for 30 minutes before washing and fixing. Then after permeabilizing and staining all samples with STV-Cy3, following the details in the text protocol, store the samples at four degrees Celsius. Finally, analyze the samples by flow cytometry.