Our protocol allows users to measure ALDH1A1 activity, a stem cell biomarker using diverse modalities ranging from flow cytometry to live cell molecular imaging. The major advantages of this approach are that probe activation involves the turnaround response characterized by a high signal to background ratio, as well as selective ALDH1A1 isoform detection. Demonstrating this procedure will be Michael Lee, Sarah Gardner, and Rodrigo Tapia Hernandez graduate students from my laboratory.
To begin, aspirate the growth media from the cultured cells of interest in eight well chamber slide. Add 500 microliters per well of serum free media supplemented with a two micromolar isoform selective fluorogenic probe or a non-reactive control probe. Incubate the slide at room temperature for 30 minutes.
After incubation, proceed to confocal microscope imaging immediately. Locate the cells at 10 x magnification. The FITC channel and transmitted light channels are required for this experiment.
Locate and focus the cells using the transmitted light channel to focus on the same Z plane for the entirety of the experiment to remove bias. Adjust the laser power and FITC gain to the appropriate setting where the signal from the control AlDeSense AM samples is minimally detectable, while still seeing signal in AlDeSense AM samples. Adjust each parameter by sliding the corresponding bar.
The settings may have to be adjusted a few times to identify the correct parameters. Once optimized, complete the rest of the experiment within that cell line using identical parameters. Snap three images per well for a total of three wells per treatment condition and save the images.
Ensure to focus using the transmitted light channel only while switching between planes and wells to avoid bias. Next, using the image processing software split the CZI file into different channels and count the total number of cells and fluorescent cells. To determine the percentage of aldehyde dehydrogenase 1A1 positive cells, divide the number of fluorescent cells by the total number of cells in each image.
Count the same way for each image to avoid confounding variables. Take out a T25 cell culture flask containing the cells maintained in the incubator. Add trypsin for cell detachment, and count the cells using an automated cell counter.
Then pellet the cells in a 15 milliliter centrifuge tube by centrifugation at 180G for five minutes at 25 degrees Celsius. Re-suspend the cells in one milliliter of a two micromolar probe or control probe solution in PBS. Rock the cells at room temperature for 60 minutes to ensure even exposure to the dye.
After the incubation period, pellet the cells by centrifugation at 180 G for five minutes at 25 degrees Celsius. Then re-suspend the cells in 0.5 milliliters of PBS. Run the cells through a 35 micrometer nylon mesh cell strainer onto a tube placed on ice to remove cell clumps that may clog the flow cytometer.
Turn the flow cytometer instrument on and run the startup protocol. Check for sheath fluid and empty waist. Run the lines with 10%bleach and water for five minutes each.
Then run quality control beads to ensure proper function. In the settings tab, select forward scatter side scatter and FITC for the fluorescence filter. Draw a forward scatter area versus a side scatter area plot for the main cell population near the center of the graph.
Next, draw a forward scatter area, versus a forward scatter width plot for the narrow horizontal bands indicating singlets. Then draw an FITC area versus a forward scatter area plot to observe the distribution of cells sorted by the isoform selective fluorogenic turn on probe. Next, draw an FITC area histogram to observe the shift in population based on FITC and determine the percentage of aldehyde dehydrogenase 1A1 positive cells.
To optimize the FITC laser power, run a sample with the probe so that the right tail of the histogram curve is near the maximum FITC area signal. The laser power optimization step may have to be repeated multiple times, but the laser power should not be altered across samples, once a setting has been designated for an experiment. Subsequently, add a sample to the sift, and run it with the control probe.
A population shift should be observable to reveal the maximum dynamic range. Run each sample for 10, 000 counts done in triplicate. After completion of sample collection, run the lines with 10%bleach and water for five minutes each, then shut down the instrument.
Process the data using flow cytometry software and gait the desired cell population. Using the rectangle gate selection, set the aldehyde dehydrogenase 1A1 negative gait, so that more than 99.5%of events in the control probe samples occur within this gate. The remaining cells will be considered aldehyde dehydrogenase 1A1 positive.
Apply the same gates to the probe sample. To quantify the number of events considered aldehyde dehydrogenase 1A1 negative and 1A1 positive. The average fold turn-ons for each cell line were determined to quantify the total aldehyde dehydrogenase 1A1 activity.
The percentage of aldehyde dehydrogenase 1A1 positive cells was determined in each cell line by tuning the laser power and gain. To minimize the signal in the control AlDeSense AM treated sample the fluorescence signal was optimized in the AlDeSense AM treated cells. By counting the number of aldehyde dehydrogenase A1A positive cells, the percentage of aldehyde dehydrogenase A1A positive cells was determined.
With the application of the isoform selective fluorogenic probe, the aldehyde dehydrogenase 1A1 positive cell population was quantified within each cell line by gating for the top 0.5%of the brightest cells within the control AlDeSense AM treated population. Analysis of the panel of ovarian cancer cells has revealed the percentage of aldehyde dehydrogenase 1A1 positive cells. From the obtained results of the tested cancer cell lines, it can be concluded that BG-1 has the lowest aldehyde dehydrogenase 1A1 activity and lowest aldehyde dehydrogenase 1A1 positive population.
Additionally, confocal imaging and flow cytometry revealed the largest percentage of aldehyde dehydrogenase 1A1 positive cells in Caov-3 cells. But the cell line's overall activity was only the third highest. Alternatively, OVCAR-3 cells contained the third highest aldehyde dehydrogenase 1A1 positive population, but exhibited the highest overall activity.
AlDeSense is a versatile and generalizable probe to identify CSCs in immortalized cell lines and in patient samples. We hope to see it as a prognostic tool in the clinical setting.
