Genetic Barcoding with Fluorescent Proteins for Multiplexed Applications

The overall goal of the following experiment is to fluorescently barcode mammalian cells to enhance multiplexed high throughput biological assays that rely on distinct fluorescent readouts. To do this, retroviral particles are used to stably express fluorescent proteins in mammalian cells, thereby barcoding them. Next fluorescent activated cell sorting is performed to obtain clonal barcoded populations.

The clonal populations are then amplified, combined and analyzed by flow cytometry to confirm that they can be distinguished from one another and therefore be used concurrently. Finally, the barcoded cell lines are transduced with tagged assay. Proteins and biological activity is monitored using fluorescent channels distinct from the barcoded channels.

The results demonstrate the utility of fluorescent bar coating of mammalian cells for multiplexed applications in a high throughput environment. The main advantage of this technique over existing methods that require careful calibration of antibody or dye is that once genetically barcoded cell lines are established, they require no further manipulation and because the protein of interest is expressed for long periods of time, they're used further for biological adaptations. Coupling fluorescent genetically barcoded cell lines with surface assays allows us to investigate biological functions or effects independently in a hydro throughput format.

In our example, we use fluorescent genetic bar coating to monitor cleavage of different viral substrates multiplexing. The assay can be further exploited to investigate the machineries involved in cleavage and for high throughput drug discovery. The day before transfection seed a 10 centimeter plate with 2.5 times 10 to the sixth Phoenix GP cells in DMEM with 10%FBS.

This packaging cell line stably expresses gag and pole proteins for retroviral particle formation in trance. On the day of the transfection, use a brightfield microscope to examine the cells. The cells must be healthy and adhere to the plate in a monolayer to be used for transfection.

Next, prepare the DNA transfection mixture to 125 microliters of serum free.DMEM. Add three micrograms of vesicular stomatitis virus envelope glycoprotein vector, P-C-I-V-S-V-G, and three micrograms of transfer vector carrying the fluorescent protein gene of interest add 15 microliters of polyetheramine transfection reagent and mix to obtain viral particles carrying different fluorescent protein markers. Perform each transfection independently with the marker of choice to do so.

Incubate each mixture at room temperature for 15 minutes following the incubation transfect by adding the mixture to the cells. Dropwise then incubate the plates at 37 degrees Celsius. Optionally, after 24 hours, replace the medium with seven milliliters of fresh medium.

Although this may improve cell health, it may decrease the viral load in the supernatant. 48 hours after transfection use a pipetter to transfer the supernatant, which contains the virus particles to a 10 milliliter syringe. Fitted with a 0.20 micromolar polytetrafluoroethylene filter to press the plunger to dispense the filtrate into two milliliter micro centrifuge tubes.

Set aside an aliquot of two milliliters for immediate use. Place the remaining viral snat and aliquots on ice and transfer them to negative 80 degrees Celsius until they are used 24 hours before transduction in each well of a six well plate seed 2.0 to 3.0 times 10 to the fifth adherent cells here 2 9 3 T cells are used the next day. To transduce the adherent cells, remove the existing media from the plate.

Add five micrograms per milliliter of poly brain to the two milliliters of the previously collected viral sine, and add the mixture carefully to the cells. Seal the plates with parfum. Then place the plates in a hanging bucket.

Centrifuge centrifuge at 1500 times G for 120 minutes at 32 degrees celsius. After the spin, incubate the cells at 37 degrees Celsius and 5%carbon dioxide. After 24 hours, replace the cells medium at least 72 hours.

Post transduction. Prepare to analyze the transduced cells by flow cytometry to quantify the percentage rate of transduction. First, using a comparable nont transduced cell line as a negative control, ensure the gates are properly set in the right channels.

Set the parameter channel voltage so that the negative population is below the value of 10 to the third on each fluorescent axis of interest. Next, set the gates for positive fluorescence as events that appear beyond that of the negative control. Then using the transduced cells, determine the percentage positive for the specific fluorescent channel.

Sort the cells into 15 milliliter conical tubes containing one milliliter of 100%FBS. Then spin down the sorted populations in a hanging bucket. Rotor centrifuge at 524 times G at 32 degrees Celsius for five minutes.

After the spin, resuspend the cells in one milliliter of fresh medium, then seed two times 10 to the six cells in two milliliters of DMEM on a six centimeter plate. At this concentration, the confluence is below 60%and will allow growth and division for at least two days. Place the cells in an incubator at 37 degrees Celsius for several days to allow time for amplification.

Clonal populations can be generated from either the originally transduced or the sorted cells. Sorting tight populations based on chosen fluorescence rather than individual clones at this stage ensures a backup population for future selection. If when needed, set the gates for sorting according to populations of interest, ensure that the gates are set at least one log scale apart To obtain distinguishable populations using an automated cell deposition unit sort single cells into 96 well plates note that there will be an average of one cell per well.

Some wells may not have any cells and others may have more than one Following. Sorting, incubate the cells at 37 degrees Celsius for at least two weeks To amplify the individual clones. Regularly examine the cells under the microscope to ensure that growing populations arise from individual cells and not from more than one.

Also ensure that the cells do not become overgrown after two weeks have passed. Screen the putative clonal populations by flow cytometry and compare them to a negative control population. True clonal populations should have very tight fluorescence patterns.

Choose the most distinctly separate populations based on average intensity and tightness of the population. Amplify them as needed or store stocks in freezing medium with 10%DMSO at negative 80 degrees Celsius for short periods of time or liquid nitrogen for longer. Next, to test whether clonal populations can be successfully used in a multiplex high throughput screening system.

Choose a set of clones with distinguishable absorption, emission spectra and or different intensities, and combine them in a single tube seed. 50, 000 of the combined cells in 200 microliters of supplemented DMEM in each well of a 96 well plate in separate tubes. Prepare negative and single color controls for use in setting parameters and compensation values at the flow cytometer.

Set the compensation values. Then analyze the sample to ensure that each of the independent established fluorescent cell lines can be distinguished. To adapt the barcoded cell lines to a biological application, first select assay elements that have been engineered such that they're coupled to a tag or fluorescent protein as a fusion or through an internal ribosome entry site.

Ires, which enables straightforward detection through western blotting flow cytometry or a microscopy. The system utilized here consists of a biological assay in which an HIV envelope protein is expressed on the surface of the cell, exposing its HA and flag tags. The assay protein is expressed in a barcoded cell that expresses TD tomato.

Cleavage of the protein can be monitored by staining with a FSE labeled Anti-Flag antibody. When the protein is not cleaved, staining is seen, but when the protein is cleaved, the green fluorescence labeling is no longer evident. To establish the biological assay in the barcoded cells, transduce them with viral particles that contain the assay elements of choice.

Add viral SUP natin to cells of choice and spin by centrifugation. Next, incubate the cells with fluorescently labeled anti HA antibodies. Then sort the barcoded cells to isolate those that contain assay elements which are positive for ha.

Finally, combined distinct barcoded cell lines. Each containing an assay bearing a different substrate. Incubate the mixture of cells with Anti-Flag and ZI conjugated antibody and analyze them by flow cytometry to determine whether cleavage of the protein has occurred to track back the assay, analyze or decode the populations in the appropriate channel where the distinct genetically barcoded cell lines can be distinguished here.

The PE channel for TD tomato then decode the nature of the substrate by analyzing in the fite channel to reveal the positive population to assay for cleavage of surface expressed proteins in the secretory pathway. Sub T one cells for fluorescently barcoded to express TD tomato and sorted into three populations at different intensities. Each of the populations was then transduced with a different retrovirus to induce the expression of wild type HIV envelope protein, the mutant HIV envelope protein or the dengue virus.

PRM boundary protein, each flanked by flag and HA tags. The transduced cells were incubated with anti HA and Anti-Flag antibodies. Anti HA staining was performed to confirm the presence of the engineered assay protein while Anti-Flag staining was performed to determine whether it was cleaved.

When unstained combined populations were analyzed by flow cytometry, clonal populations were distinguishable based on the TD tomato barcode, but indistinguishable the fite channel. In contrast, when the same populations were stained with flag fite antibody, one population was positive for flagg. Based on barcoding, this population can easily be identified as the population bearing the mutant HIV envelope substrate it.

Now, you should have a very good understanding on how to utilize retro valve technology together with flow cytometry. In order to establish fluorescent genetically barcoded cell lines, four biological applications in a multiplex format, The ability to couple fluorescent genetically barcoded cells with cell-based assays in a multiplex format dramatically enhances high throughput capabilities. After the cell lines are generated, it's important to freeze them down.

Then periodically thaw them, reanalyze em, and frees them back down. While generating fluorescent genetically barcoded cell lines might be time consuming at first, once established, they dramatically enhance repeatability and robustness of future applications.