The overall goal of this procedure is to study the response of human immune cells to respiratory syncytial virus or RSV infection. This is accomplished by first culturing an infectious RSV stock on helo cells. In the second step, the cultured RSV stock is purified by ultracentrifugation.
Next primary human blood immune cells are isolated by density centrifugation. In the final step, the isolated immune cells are stimulated with the virus. Ultimately, the cellular immune response to RSV infection can be assessed by E iza QPCR or flow cytometry.
This technique is a valuable addition to study respiratory syn situ virus infections. RSV is highly adaptive to humans. Therefore, it's important to use human immune cells to study the inflammatory pathways involved in the pathogenesis of the virus.
Although the virus used in this video is RSV, this method can also easily be adapted to study other viruses such as influenza virus or rhinovirus. Begin by warming a vial of human RSV seating stock and a 37 degree Celsius water bath. When the virus has thawed dilute the stock in four milliliter aliquots of infection media per flask.
Keeping the MOI at 0.1 or below, add the virus to the heli cells and as low a total volume as possible without causing the cells to dry out. And then incubate the cultures for two to four hours at 37 degrees Celsius and 5%CO2, swirling the flask every 15 minutes to redistribute the medium. Next, remove the inoculum and wash the cells once with PBS.
Then add 15 milliliters of culture medium to the flasks and incubate them for three to five days under the same culture conditions. Observe the cells once a day every day under normal bright field microscopy for virus induced cytopathological changes. When these changes are observed, scrape the cells to harvest the RSV and then collect the suspension in a sterile 50 milliliter tube, cortex the cells, and then centrifuge them for 10 minutes at 1800 Gs and room temperature to pellet the cell debris.
RSV does not pellet at this speed, and therefore the supernatant can be stored at minus 80 degrees Celsius as seeding stock in one milliliter. Aliquots for future use always snap freeze RSV and liquid nitrogen to prevent the loss of infectious virus particles. To purify RSV begin by placing a sterile centrifuge tube in each bucket of an ultracentrifuge.
Then add five milliliters of freshly prepared filter sterilized sucrose solution to the bottom of each tube. Next, very slowly, add 30 milliliters of the RSV solution on top of the sucrose solution, taking care not to disturb the sucrose solution and to keep the layers distinct Then in a flow cabinet to maintain sterility, carefully balance the six ultracentrifugation buckets with sterile PBS to within 0.1 grams of each other, including the caps. Spin down the virus for 1.5 hours at 20, 000 RCF, and four degrees Celsius.
And then when the spin is completed, use sterile forcep to carefully remove the centrifuge tubes from the buckets. The virus particles will have passed through the sucrose layer to form a pellet. Tip the centrifuge tubes into a viral waste flask and let the tube drain upside down on a sterile surface.
Do not let the pellet completely dry out. Next carefully, let one milliliter of HPSS run over the pellet and tip the centrifuge tube again to drain the HPSS. Then turn the tube upside down again, being careful to keep the pellet from drying out.
Now tri rate the pellet 40 times in another milliliter of HBSS taking care to avoid bubble formation as this can destroy the virus. Then snap freeze aliquots of the virus in liquid nitrogen. Store the virus at negative 80 degrees Celsius until quantification and further use After obtaining blood in 10 milliliter EDTA tubes from healthy volunteers dilute the blood with room temperature PBS up to two times to a maximum of 35 milliliters of diluted blood in a 50 milliliter tube.
Next, attach a sterile lure lock needle to a sterile 50 milliliter syringe and then aspirate density gradient media into the syringe. Place the needle on the bottom of the tube of diluted blood and carefully pipette 15 milliliters of density gradient media under the blood, keeping a clean demarcation between the density gradient and the blood. Now separate the cells for 20 minutes at 800 GS in room temperature at the maximum acceleration with the break off After the separation, the erythrocytes are found in the pellet at the bottom of the tube with a silvery layer of neutrophils on top.
The clear layer above the pellet contains the density gradient media and the thin layer between the density gradient media and the blood plasma layer on top contains the pbmc. Use a pastier pipette to carefully collect the pbmc, then transfer the cells to a new 50 milliliter tube on ice. Add ice cold PBS to a volume of 50 milliliters and pellet the cells when the spin is completed.
Aspirate the supernatant and wash the pellet in 50 milliliters of ice cold PBS two time after the second wash, add one milliliter of ice cold medium to the pellet, count the cells and adjust the cell concentration to five times 10 to the six pbmc per milliliter. To stimulate the pbmc with live RSV first, add 100 microliters of pbmc to each well of a 96 well round bottom plate. Then add 50 microliters of two freshly diluted stimuli to each.
Well incubate the plate for 24 hours at 37 degrees Celsius and 5%CO2, and then centrifuge the plate, collect the supernatant for later analysis by Eliza. The stimulated pbmc in the pellet can be analyzed by QPCR or stained for flow cytometry. When performing A-P-B-M-C stimulation experiment, the stimuli fraction method is suggested with this method.
The conditions are divided into standard stimuli conditions such as RPMI or human RSV and test fraction conditions such as mial, dipeptide or LPS. Importantly, a negative control such as RPMI alone and a positive control such as LPS are always included in each experiment after 24 hours of stimulation with RSV different PBMC populations exhibit a dose dependent infection rate and a difference in RSV infection efficiency After 24 hours of stimulation with virus qPCR R analysis of the P BMCs for the expression of TNF alpha or interferon gamma demonstrates that RSV induces robust upregulation of the transcription of these two inflammatory cytokines. Conversely, stimulation with miam dipeptide or LPS result in only a weak mRNA expression.
When P BMCs are stimulated with both RSV and MIAL dipeptide together, a strong synergistic upregulation is observed. Stimulated pbmc were also analyzed for their production of TNF Alpha or Interferon gamma by Eliza. In contrast to the observations at the transcriptional level, RSV is a poor inducer of TNF alpha and interferon gamma protein secretion by p BMCs stimulation with miam dipeptide results in the production of low amounts of TNF alpha and no interferon gamma.
Similar to the transcriptional analyses, both TNF alpha and interferon gamma Exhibit A synergistic upregulation when co stimulated with RSV and MIAM dipeptide as a control to see whether cytokine induction is dependent on RSV replication. P BMCs were stimulated with live or beta propal, lactone inactivated RSV as illustrated in the graphs both live and inactivated RSV induced PRO-inflammatory cytokine production. Don't forget that experiments with RSV must be performed in a biological safety cabinet in a biosafety level dual laboratory.
Moreover, while attempting this method is crucial to remember to use endotoxin free reagents and materials throughout the entire procedure, After watching this video, you should have a good understanding of how to stimulate human pbmc with RSV and of several assays that can be used to analyze the immune response.I.
