Real-time Live-cell Flow Cytometry to Investigate Calcium Influx, Pore Formation, and Phagocytosis by P2X7 Receptors in Adult Neural Progenitor Cells

P2X7 receptors are extremely versatile and function differently depending on the amount of agonist present. In this way, P2X7 has different functions and can regulate the physiology of many different systems. This protocol allows for investigation of the three major functions of the P2X7 receptor.

It is a rapid, reproducible and quantifiable method for understanding its function. Our focus in on how P2X7 receptors can modulate in neural progenitor cells. However this method can be easily adapted to suit any cell type.

Students who have read enough literature before trying this procedure can learn that in just one day and be able to run this all alone after two or three tries. Begin this procedure by obtaining neural progenitor cells from the subventricular zone and hippocampus of adult mice as described in the text protocol. Maintain cultures at 37 degrees Celsius with 5%carbon dioxide.

Neurospheres should form after seven to 10 days is the passage zero subventricular zone culture and after 15 to 20 days in the hippocampal culture. Following the initial passage zero culture phase, passage every seven to 10 days as necessary using a biological safety cabinet and standard tissue culture practices. Passage the spheres when they reach 150 to 200 microns in diameter.

Collect the spheres and medium in a 15 milliliter tube and spin at a low speed for two minutes. Remove the medium and incubate the cells with dissociation reagent for seven to 10 minutes at 37 degrees Celsius depending on the size of the spheres. Finally, count the cells using a hemocytometer or automatic cell counter.

We suspend the single cells in one milliliter of calcium-free sodium medium. Then load the cells with two ninigrams per milliliter of calcium indicator dye and 10 microliters of 5%pluronic acid. Incubate the cells for 30 minutes at 37 degrees Celsius.

Wash the cells by adding three to five milliliters of calcium-free sodium medium and gently centrifuging. Remove the supernatant and re-suspend the cells in calcium-free sodium medium, washing a second time. Re-suspend the cells in one milliliter of calcium-free sodium medium.

Then place the cells on ice and allow them to de-esterify for 30 minutes. Wash the cells once more by adding three to five milliliters of potassium medium and centrifuging as before. After re-suspending the cells in potassium medium, eliquate them into fluorescence-activated cell sorting or FACS tubes at a concentration of one million cells per 500 microliters per FACS tube.

Place the FACS tubes on ice until the cells are ready to be analyzed. Do not leave the cells on ice for an extended period but begin the assay as soon as possible. For some samples, pre-incubate the cells with P2X7 receptor-specific inhibitor as described in the text protocol.

A few minutes prior to running the first sample, add calcium chloride to a final concentration of one millimolar and place the tube in a 37 degree Celsius water bath to recover. Drop a clean small magnetic stirrer into the FACS tube and position the tube in the time module which is linked to a circulating 37 degree Celsius water bath to control the sample temperature. Select a low stirring speed to ensure movement of the sample without introducing a vortex effect.

Place the water jug and tube adapter onto the sample platform and close the lever arm of the FACS machine. Initiate sample acquisition and run the sample for three minutes at around 1, 000 events per second. At the 40 second mark, quickly remove the tube and add the P2X7 agonist.

Either one millimolar ATP or 300 micromolar BzATP. Then replace the tube to continue the acquisition. While the first sample is recording, prepare the second sample with calcium chloride.

Place it in 37 degrees Celsius to allow sufficient time for the cells to warm up prior to analysis. Once the first sample has finished, clean the intake by running a water sample. Then the acquisition of the second sample can begin as before.

Always clean the intake between samples. Create a single-cell suspension as before and save a few milliliters of the conditioned medium. Use the medium to re-suspend the cells at a concentration of one million cells per 100 microliters per FACS tube and place the cells on ice until ready.

Prior to running the assay, add 900 microliters of potassium medium for a final volume of one milliliter. Place the tubes in a 37 degree Celsius water bath for 10 minutes to recover. If applicable, pre-incubate the cells with treatments including the P2X7-specific inhibitors.

Immediately prior to running the assay, add 25 micromolar ethidium bromide to the FACS tube. Then add the magnetic stirrer, place the tube on the FACS machine and begin the acquisition as before. To induce the formation of pores in the cell membrane, add one millimolar ATP or 100 micromolar BzATP 40 seconds after the start of the acquisition.

Run the samples at around 1, 000 events per second for six minutes. While the first sample is running, take the second sample from the ice and place it in the 37 degree Celsius water bath to allow sufficient time for the cells to recover prior to analysis. Once the first sample acquisition has finished and the intake has been cleaned, the second sample can be placed on the machine to begin recording.

We suspend the single cells in conditioned medium and eliquate them into FACS tubes at a concentration of at least one million cells per 100 microliters per FACS tube. Dilute the cells to a final concentration of one million cells per milliliter with sodium medium and place the cells on ice until the analysis is performed. Use one micron wide G-beads as phagocytic targets for real time phagocytosis assays.

Prior to running the first sample, transfer the cells to a 37 degree Celsius water bath and incubate them for approximately seven to 10 minutes to allow the cells to recover. Add any treatments requiring pre-incubation to their respective tubes including ATP, oxidized ATP, cytokelisine D and 4%paraformaldehyde. No pre-incubation is required for 5%human serum.

If treatments are added at approximately the same time, the samples can be run consecutively while others continue to incubate because their incubation times vary. Place the sample on the cytometer with the magnetic stirrer and initiate sample acquisition as before. Remove the sample tube from the machine 15 to 20 seconds after the start of the acquisition and add five microliters of undiluted YG beads.

Return the sample FACS tube to the instrument and continue the acquisition. Run the samples for seven to eight minutes at around 1, 000 events per second. While the first sample is running, take the second sample from the ice and place it in a 37 degree Celsius water bath to allow sufficient time for the cells to recover prior to analysis.

Once the first sample in finished and the intake has been cleaned, begin acquisition on the second sample. When plotted over time, calcium influx was generally similar in the hippocampal and subventricular zone neural progenitor cells. Agonists were added at the 42nd mark.

BzATP rapidly activates P2X7 receptors, opening the ion channel and allowing calcium influx which binds to fluo-8 and fluoresces. ATP application generally results in a more gradual calcium influx. It has a lower affinity to P2X7 when compared to BzATP and will also result in G-protein coupled receptor activation.

Following the application of the agonist's ATP and BzATP, time-resolve flow cytometry captures the ethidium bromide entering the cells through transmembrane pores in real time. This effect was attenuated by the P2X7-specific inhibitor. ATP concentration response assays illustrate the effects of agonist concentration on P2X7 pore formation using change in ethidium bromide fluorescence over time.

Here P2X7 receptor-involved phagocytosis by hippocampal and subventricular zone neural progenitor cells is show in real time. Uninhibited phagocytosis levels of YG latex beads were established as the positive control. ATP inhibited the phagocytosis of YG beads to the same extent as the non-specific inhibitors, namely paraformaldehyde fixation and the actin polymerization inhibitor cytokelisine D.5%serum abolished all innate phagocytosis.

Maintaining a healthy cell population and reducing time on ice is vital for gaining reproducible results. Variability can be minimized by using the same batch of ATP for the entire cell assay. Our timed result of flow cytometry demonstrated here is the only message which can continue select quantities for flows and changes in a targeted sub-population.

Alternative method is a fluorescent microscope and a fluorescent platter reader. However, this method cannot continuously measure fluorescent changes because of the fluorescent affinity program. P2X7 is uniquely multi-modal.

Discovering its expression on a cell of interest open up a series of unique questions for investigators to explore.