A Flow Cytometry-Based Cell Surface Protein Binding Assay for Assessing Selectivity and Specificity of an Anticancer Aptamer

This protocol will help researchers new to working with aptamers to be able to use them in their projects and also to understand the individual little steps that are required to complete the protocol. Aptamers are very versatile, and this protocol demonstrates how easy and simple they are in an immunophenotyping assay. Aptamers can be used for both diagnostic and therapeutic applications.

This protocol is the first steps undertaken to confirm their specificity and sensitivity. While this particular assay demonstrates staining of one cell surface receptor, aptamers can very easily be used for multi-parametric flow cytometry, providing a high level of phenotypic knowledge. The important aspects of working with aptamers are ensuring that they are folded correctly prior to use, and also that the buffer and the ionic strength conditions are the same as when they were selected.

After collecting and discarding the media in the flask, add three milliliters of PBS and spread it over the cells. After adding one milliliter of 0.25%of Trypsin-EDTA to each flask, incubate for five to 10 minutes at 37 degrees Celsius and visualize the detachment of cells under a microscope. Next, add one milliliter of complete medium to the cells and pipette the cells up and down to make a single cell suspension.

Pipette the cells into an appropriate tube and centrifuge at 200 G for five minutes. After discarding the supernatant, resuspend the cells in one milliliter of fresh medium and count the cells using trypan blue staining by diluting certain volume of cell suspension with trypan blue. Distribute approximately 15 microliters of the mixture between a hemocytometer and a cover glass to count the cells.

Collect the required number of cells, making sure to have a hundred thousand cells per test sample. Then incubate the cells at 37 degrees Celsius for two hours to allow the stabilization of the protein of interest on the cell membrane following enzymatic detachment. Following the two hours incubation, centrifuge the cells at 500 G for five minutes.

After discarding the supernatant, resuspend the cells in 500 microliters of blocking buffer. Incubate the cells at four degrees Celsius for 30 minutes with intermittent mixing. During this 30 minute incubation, perform aptamer folding by preparing twice the concentrations of aptamers as described in the manuscript.

Then mix and incubate the aptamers with the thermocycler machine according to the required folding conditions while ensuring to protect the aptamers from light. Following the 30 minutes incubation, centrifuge the cells as demonstrated previously and remove the supernatant. Then add one milliliter of wash buffer and centrifuge the cells again.

After removing the supernatant, resuspend the cells in a suitable volume of the binding buffer. Next, pipette 50 microliters of the resuspended cells into each well of an ice cold 96 well black plate. Then pipette 50 microliters of the aptamers onto a 50 microliters volume of cells, mix and incubate in darkness at four degrees Celsius for 30 minutes.

After centrifuge and removing the supernatant, carefully resuspend the pellet in wash buffer and again centrifuge at 500 G for five minutes. Then resuspend in 100 microliters of wash buffer for flow cytometric analysis while repeating the wash step twice. First, turn on the flow cytometer and then the computer.

Next, open the flow cytometry analysis software, log into the program, and under the cytometer tab run fluid startup. To create a new experiment, under the experiment tab, click new folder, and name the folder slash experiment appropriately. Click on the new folder to highlight it.

Then under the experiment tab again, click new experiment and name the experiment appropriately. To add the first sample slash specimen, under the experiment tab click new specimen and name the specimen appropriately. To add a tube sample, highlight the specimen and under the experiment tab, click new tube.

Then add the appropriate number of tubes and name. To prepare the required graphs, under a worksheet tab, open a new worksheet. Once the new worksheet window pops up, prepare a dot blot graph of side scatter versus forward scatter to select the population of interest.

Prepare a dot blot graph of forward scatter height versus forward scatter area to select the single cell population. Next, prepare a histogram of the number of events against the fluorophore of interest. Ensure that the acquisition dashboard for controlling the sample acquisition, inspector and cytometer to adjust voltage parameters as well as the worksheet with all the graphs is open before starting flow cytometry.

First, run the untreated cells. Make sure the arrow pointing to the tube is green. If this arrow is not green, click on the arrow to make it green.

Using up pipette, transfer each sample from the 96 well black plate to a flow cytometry tube. On the acquisition dashboard, choose an appropriate number of events to record, change the flow rate to low, and click Acquire Data. Adjust the voltage for the FSC and SCC parameters, ensuring that the cell population is centralized within the dot plot and that no cells are touching either axis of the graph to avoid losing the cells of interest.

Define the first gate by identifying and selecting the population of interest in a forward and side scattered density plot while excluding the debris, which constitutes the population with the lowest forward scatter signal. Define the second gate by excluding doublet cell populations, as doublet cells considerably affect the results and conclusions. Increase the acquisition speed to medium or high to analyze the samples faster, but do not exceed more than 200 to 300 events per second.

Then click record data. After adjusting the voltage and gating and recording the data, take out the sample and click Next Tube. Insert the next sample and repeat recording data for all control and test samples.

Once all the data are collected, wash the flow cytometer by running three tubes of 50%bleach, FACSRinse and ultrapure water, each for five minutes at a high flow rate. Then from the cytometer dropdown menu, click fluidics shut down. Export the result as fsc files to a USB drive to transfer.

Analyze them using the analysis software by pressing the new button to create a new document and window to handle the analysis, and drag the sample files into the new window. Double click to open the unstained sample. To gate the single cell population, choose the P1 population and double click on the P1 population to create an FSCH versus FSCA graph.

Double click on the gated single cells to create a histogram of events against the used fluorochrome. In the original window select P1 and single cells and drag them to all samples so that all samples now contain the same gating. Next, click on the layout editor button to open the layouts window and drag two samples over one another to create overlay histogram.

In EpCAM positive MDA-MB-231 cells, overlaying the histograms representing cells treated with TEPP and TENN shows that the TEPP treated cells are shifted to the right, compared to the TENN treated cells demonstrating that the binding of the TEPP aptamer occurs to the protein of interest. In negative control, HEK 293T cells overlaying histograms of TEPP and TENN do not reflect any shift indicating that in EpCAM expressing cells TEPP is the EpCAM aptamer attached to its receptor. And furthermore, no binding was observed in EpCAM negative cells, which confirms the selectivity and specificity of the developed aptamer.

Ensure the aptamers are protected from light, the aptamers and cells are mixed on the plate, and the correct voltages are set on the flow cytometer. This is highly dependent on future applications for the aptamers. For example, we are focused on drug delivery.

So the next step for us would be to assess if the aptamers are internalized using fluorescent microscopy. This protocol has demonstrated how easy it is for aptamers to replace monoclonal and polyclonal antibodies in immunophenotyping assays.