High-throughput Titration of Luciferase-expressing Recombinant Viruses

The overall goal of the following experiment is to estimate viral titers of a luciferase tagged virus using a high throughput quantification method. This is achieved by transferring the supernatant of the samples to be titered, as well as a viral standard curve onto a permissive cell line to allow for luciferase expression. As a second step, RESA zurin can be added to the original samples, which will be used to assess cell viability.

Next, following an appropriate incubation time, luciferian is added to the cells in order to quantify by luminescence. The results show the amount of virus in the sample based on luciferase expression. The main advantage of this technique over existing methods like plaque tittering, is that a large number of samples can be processed quickly and simultaneously.

This method can also be extended to non clacking luciferase expressing viruses. Since it is not dependent on plaque formation, A readout of cell cytotoxicity can easily be incorporated into the workflow and may be useful in the context of a drug screen. Demonstrating the procedure will be Vanessa Ramia and Colin three graduate students from my laboratory First perform infections using VSP Delta 51 expressing Firefly Luciferase in 96.

Well, tissue culture plates supernatants from these plates will be titered after an appropriate incubation time 24 hours prior to titering. Prepare a suspension of Vero cells at a concentration of 2.5 times 10 to the fifth cells per milliliter in DMEM containing 10%FBS 30 millimolar heaps and 1%penicillin streptomycin C, 2.5 times 10 to the fourth cells in 96. Well white, solid flat bottom plates using a microplate dispenser to prepare the cell suspension, clean a microplate dispenser cassette by flushing the tubing with 50 milliliters of sterile water.

Then fill the microplate dispenser lines with cell suspension, allowing five milliliters of the cell suspension to flow through. Select a program that will dispense 100 microliters into each well of a white walled 96 well plate dispense and repeat as necessary for additional plates. Also seed a few wells in a 96 well clear white bottom plate for verification of cell health and density.

When finished, flush the cells back into the original container. Clean the cassette by running 50 milliliters of 70%ethanol, followed by 50 milliliters of warm sterile water through the tubing. Following this, incubate the cells for 24 hours at 37 degrees Celsius in a humidified 5%carbon dioxide incubator.

Prepare a standard curve of VSV Delta 51 in serum free DMEM such that the final concentration of platforming units per milliliter after transfer onto Vero cells is as follows. Prepare 50 microliters of each concentration per plate of Vero cells, plus an extra 10%The standard curve can be transferred into a 96 deep well plate. Next, check the Vero cells plated in the clear bottom 96 well plate under a light microscope to confirm that monolayers are at least 95%confluent trans transfer.

25 microliters of sample supernatant onto the Vero cells seated in the white walled plates using a 12 channel multichannel pipetter at 25 microliters of each dilution of the standard curve to the Vero cells in the two designated columns. Centrifuge the plates for five minutes at 430 Gs at room temperature. Then incubate the plates for five hours at 37 degrees Celsius in a humidified 5%carbon dioxide incubator.

At this point, add zurin in an amount equal to 10%of the volume in each well of the 96 well plate of samples containing virus and cells. After two to four hours, read and record the signal with a fluorescence plate reader using 530 to 560 for excitation and 590 for emission report. Cell viability for the sample according to the following formula.

30 minutes before the end of the five hour incubation, prepare the Lucifer to obtain a two milligram per milliliter solution in sterile phosphate buffered saline. Protect the solution from light after the five hour incubation period at 25 microliters of Lucifer to each well of Vero cells in the white solid plates with an automated dispenser integrated in the luminometer. Read the plates with the following parameters.

Shake for five seconds and wait for 30 seconds. Read luminescence at an appropriate fixed sensitivity slash exposure value, then record and save the quantified bioluminescent intensity. If an automated dispenser was used to add luciferian, perch the luciferian and prime the lines with sterile water or accurate results, solve the nonlinear regression to generate a hill equation from the standard curves.

Apply this equation to the titered samples to calculate viral expression units. The results of a typical standard curve of VSV Delta 51 are shown here, or parameter nonlinear regression analysis generated a hail plot from which unknown input forming units can be interpolated. Viral titers obtained by standard plaque assay on Vero cells were compared with calculated viral titers from the same samples.

Tittered using the high throughput luciferase assay samples originated from an experiment where various chemicals were used to enhance the replication and spread of VSV Delta 51 and 7 8 6 0 cells. The linear correlation between titers and viral expression units with an R squared of 0.8083 and Pearsons R score of 0.899 is pictured here. Typical cell cytotoxicity data obtained from a metabolic assay is shown here for 7, 8 6 0 cells treated with chemicals prior to infection.

With VSV Delta 51, typical standard curves obtained with herpes simplex virus, vaccinia virus, and adeno-associated virus, all expressing firefly luciferase are displayed here. Once mastered, this technique can be used to conduct high throughput screens of compound libraries in combination with your virus of interest to generate titers and cytotoxicity data. While attempting this procedure, it's important to remember to properly dilute the standard curve as this is critical for interpolating viral expression units.

Don't forget that working with live viruses can be extremely hazardous and precautions such as working in a biological safety cabinet and wearing the appropriate personal protective equipment should always be taken while performing this procedure. After watching this video, you should have a good understanding of how to estimate viral titers using a high throughput quantification method based on the expression of a luciferase transgene measurement of bioluminescence in interpolation of unknown viral expression units. From a standard curve sample, cytotoxicity can be simultaneously determined by an appropriate viability assay.