Measuring Attachment and Internalization of Influenza A Virus in A549 Cells by Flow Cytometry

The overall goal of this procedure is to measure attachment and internalization of influenza. A virus particles into a 5 49 lung epithelial cells. This is accomplished by first cold binding by a ated influenza, a virus to a 5 49 cells, which allows attachment but prevents uptake of viral particles.

Next, the virus is allowed to internalize by shifting the temperature to 37 degrees Celsius. Then a blocking step in which unlabeled strp Aden is bound to cell surface. Attached viruses is performed in order to discriminate between attached and internalized viral particles.

Finally, the cell associated biotinylated virus is incubated with CY three labeled streptavidin and flow cytometry is used to detect the stained viruses. Ultimately, the relative amount of attached and internalized viruses is calculated. Though this method can provide insight into the relative amount of influenza, a virus that attaches and internalizes into a 5 0 9 cells.

It can also be applied to other systems using different cell lines or other viruses After generating biotinylated virus and determining the required amounts of the labeled virus strep din and SI three labeled strep din according to the text protocol, prepare four deep well tubes each containing 200, 000, A 5 49 cells labeled the tubes as zero minutes. Zero minutes, plus STV, 30 minutes and 30 minutes plus STV. Next, spin the tubes.

Remove the supernatant and use 200 microliters of PBS to resuspend the pellets. Then after spinning the tubes again, use 200 microliters of a 5 49 standard growth medium to resuspend the cells incubate at 37 degrees Celsius for 30 minutes following the incubation centrifuge the samples and resuspend the pellets in 200 microliters of PBS 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 re suspend 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.

After spinning, use 100 microliters of 3.7%paraform aldehyde or PFA to resuspend the cell pellet. Incubate at room temperature for 10 minutes before spinning and using PBS to wash the cells twice following fixation. Store at four degrees Celsius until all samples have been processed.

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 azi 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 door the samples at four degrees Celsius for the 30 minute samples. After spinning and removing the supernat, use PBS 2%BSA to dissolve the samples.

Incubate at 37 degrees Celsius for 30 minutes before washing with PBS and fixing with PFA. Then store the samples at four degrees Celsius for the 30 minutes plus STV samples. After pelleting, we suspend 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 azi in PBS incubate on ICE for 30 minutes before washing and fixing. Then after permeable and staining all samples with STV SI three, following the details in the text protocol, stored the samples at four degrees Celsius. Finally, analyze the samples by flow cytometry.

Determine the percentage of CY three positive cells in each sample. Calculate the percentage of attached virus and the relative amount of internalized virus. According to the text protocol as shown here in the zero minutes sample, ated virus is cold bound to target cells as visualized by St.V.SI three staining.

When STV is applied as a blocking step virus at the cell surface can no longer be detected by S St.V.SI three staining strongly reducing the signal. When the temperature is raised to 37 degrees Celsius, attached virus is taken up into cells. Internalized virus is not sensitive to blocking with unlabeled STV resulting in a less dramatic drop in signal intensity in the 30 minutes plus STV samples shown.

Here are the percentages of SI three positive cells for all experimental conditions described in this video. Mock infected cells give the background signal intensity for the experiment and act as a negative control for virus attachment. Another control neuraminidase strongly abrogates IAV attachment by 90%compared to untreated cells.

Sodium azi, which depletes a TP, thus inhibiting endocytosis, inhibited the internalization of virus particles both during and after infection, as shown here following sodium azi treatment cells incubated for 30 minutes at 37 degrees Celsius showed a dramatic drop in internalization compared to untreated cells incubated under the same conditions. After watching this video, you should have a good understanding of how to measure attachment and internalization of influenza, a virus in a 5 0 9 cells. This protocol allows for convenient readout by flow cytometry and can be easily adapted to other experimental settings.