The authors would like to thank Maria Kasherman and Xin Huang for their contributions to this research. This work was supported by grants from the Rebecca L. Cooper Medical Research Foundation to M.W., T.C.L., and M.L., and to T.C.L. from the National Health and Medical Research Council (NHMRC) of Australia (571100 and 1048082) and the Baxter Charitable Foundation (Sydney, Australia). B.G. was supported by the Australian Research Council (ARC) Future Fellowship (FT120100581), NHMRC Project Grants (1048082, 1061419, and 1120095) and the Victorian Government&#8217;s Operational Infrastructure Support Grant to the Florey Institute. M.L. was supported by a Charles D. Kelman, M.D. Postdoctoral Award (2010) from the International Retinal Research Foundation (USA).

Animals were treated in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes and approved for use by the Griffith University Animal Ethics committee.
1. Neurosphere Culture of Neural Progenitor Cells from the Adult SVZ and Hippocampus
NOTE: The dissection protocol presented here is based on work by Walker and Kempermann, and a detailed protocol for the dissection of neural progenitor cells from adult mice is available elsewhere14. Culture conditions have been modified from Babu and colleagues15. Adult female C57BL/6 mice aged 8&#8211;12 weeks were used.
<ol>
	<li>In a biological safety cabinet, prepare the culture medium (neural basal medium) supplemented with neural stem cell proliferation supplement, 2 mM glutamine, 20 ng/mL epidermal growth factor (EGF), 10 ng/mL basic fibroblast growth factor (bFGF), and 2 &#181;g/mL heparin.</li>
	<li>Euthanize two mice by CO2 inhalation. Alternatively, anesthetize the mice according to institutional guidelines and immediately euthanize them by cervical dislocation. Spray their heads with 70% ethanol and decapitate them. Transfer each head to a sterile tube containing Hank&#8217;s balanced salt solution (HBSS) with 1x penicillin/streptomycin solution (P/S).</li>
	<li>Perform dissections in a laminar flow hood under a dissection microscope.</li>
	<li>Using forceps and dissection scissors, remove tissue and bone to expose the brain, and transfer it to a sterile dish containing HBSS with P/S.</li>
	<li>Position the brain ventral side up and use a scalpel blade to make a complete coronal incision across the optic chiasm. Hold the brain steady with the forceps and use one swift, clean, downward motion. Avoid sawing to minimize cell death and to help maintain tissue structure. 
	NOTE: If not held securely, the brain may tilt forward during the cut, compromising the number of progenitor cells obtained from the SVZ.</li>
	<li>Isolate the SVZ from the rostral half of the brain.
	<ol>
		<li>Locate both ventricles, separated by the septum, with the corpus callosum forming a white bridge above them.</li>
		<li>Use forceps to remove the septum separating the two ventricles and isolate the lateral walls of the anterior lateral ventricles. Do this by cutting away above, below, to the sides, and at the front with fine dissection scissors. There will remain just a small cup-like shape.</li>
		<li>Prepare a clean Petri dish lid with a few drops of HBSS in the middle and transfer the SVZ to the liquid. Do not allow the tissue to dry or to touch a dry surface. Stand to the side while dissecting the hippocampi.</li>
	</ol>
	</li>
	<li>Isolate the hippocampi from the caudal half of the brain.
	<ol>
		<li>Make a midline cut between the hemispheres to sever the corpus callosum. Use the lateral ventricles as a guide and gently unfold the cortex to expose the hippocampus. Once the cortex has been unrolled, the hippocampus can be seen as a dense, white, curved structure.</li>
		<li>Use fine dissection scissors or forceps to isolate the hippocampus from the neighboring tissue.</li>
		<li>With forceps, remove excess white matter, blood vessels, and any membranous tissue covering the hippocampus.</li>
		<li>Prepare a clean Petri dish lid prepared with a few drops of HBSS and transfer the hippocampus to the liquid. Repeat for the other hemisphere.</li>
	</ol>
	</li>
	<li>Once the hippocampus and the SVZ from two mice have been isolated and transferred to their respective Petri dish lids, mechanically dice the tissue using a scalpel blade. Chop the tissue in one Petri dish lid for approximately 1 min, rotating every 10 s until the tissue appears smooth and only fine pieces remain. Take a clean scalpel blade and repeat for the other dish, dicing the tissue for 1 min.</li>
	<li>Using a 1 mL pipette, transfer all the tissue from each Petri dish lid to separate tubes containing 1 mL of 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA). Use one tube for the SVZ and one tube for the hippocampus.
	<ol>
		<li>Do this by first pipetting about half of the trypsin-EDTA to the tissue in the dish to minimize air bubbles and to prevent the tissue coming into contact with the dry pipette tip as it is transferred to the tube. Rinse the dish and the scalpel blade with the rest of the trypsin-EDTA to collect as much tissue as possible and add it to the tube.</li>
	</ol>
	</li>
	<li>Incubate the tissue with the trypsin in a 37 &#176;C water bath for 30 min, agitating the tube every 10 min to properly dissociate the tissue.</li>
	<li>Triturate the tissue using a fire-polished glass Pasteur pipette to create a single cell suspension. Take care not to overtriturate as this will cause excessive cell lysis. This is a critical step for optimal tissue dissociation.</li>
	<li>Observe the tube contents during the trituration process and stop at the earliest signs that the majority of tissue clumps have been removed. The trypsin solution should become slightly cloudy when the cells have gone into single-cell suspension, although some clumps may remain. 
	NOTE: As an indication, passing the suspension up and down 10x&#8211;15x is adequate.</li>
	<li>Add culture medium up to 5 mL to neutralize the trypsin, and spin at 300 x g for 3 min. Wash a further 2x with HBSS and pass the medium through a 70 &#181;m cell strainer to remove any tissue clumps.</li>
	<li>Transfer all the cells in 15 mL of medium to a T75 cell culture flask. 
	NOTE: Spheres should form in the passage zero (P0) SVZ culture after 7&#8211;10 days and after 15&#8211;20 days in the hippocampal culture. Refrain from washing or feeding the cells during this time to maximize the number of neural progenitor cells in culture. If more progenitors are required, or to decrease the P0 incubation time, it is possible to increase the number of mice sacrificed.</li>
	<li>Maintain cultures at 37 &#176;C with 5% CO2 and, following the initial P0 culture phase, passage every 7&#8211;10 days as necessary, using a biological safety cabinet and standard tissue culture practices. 
	NOTE: The P0 hippocampal culture should generate enough spheres to passage into a T25 flask; the SVZ culture will generate many more spheres and can be passaged into a T75. If the P0 hippocampal spheres have adhered, use a 200 &#181;L pipette to gently lift the sphere.</li>
	<li>Passage the spheres when they reach 150 to 200 &#181;m in diameter. Collect the spheres and medium in a 15 mL tube and allow the spheres to settle by gravity for approximately 5 min. Alternatively, spin at a low speed (100 x g) for 2 min.</li>
	<li>Remove the medium and dissociate the cells with dissociation reagent for 7&#8211;10 min, depending on the size of the spheres. 
	NOTE: The dissociation reagent used will have significant effects on the outcome of the culture, so please refer to the Table of Materials for specifics.</li>
	<li>Wash the cells by adding 5 mL of HBSS to the cell solution and centrifuge at 300 x g for 3 min. Remove the supernatant, resuspend the cells in culture medium, and seed at approximately 150,000 cells in 5 mL of medium in a T25 cell culture flask or equivalent. Maintain the cultures at 37 &#176;C and 5% CO2.</li>
	<li>Confirm neural progenitor cell status by identifying the expression of stem cell markers such as Sox2 and ASCL1 before proceeding to any downstream protocol9. 
	NOTE: This can be done by the researcher&#8217;s preferred method (e.g., immunochemistry by microscopy, flow cytometry or western blot, or by using quantitative polymerase chain reaction (qPCR)).</li>
</ol>
2. Preparation of a Single-cell Suspension for Analysis by Flow Cytometry
<ol>
	<li>Prepare the required media. These include the following.
	<ol>
		<li>Prepare Na+ medium containing 145 mM NaCl, 5 mM KOH, 10 mM HEPES, 5 mM D-glucose, 0.1% bovine serum albumin (BSA), and 0.1 mM CaCl2. This medium is also used in a calcium-free form, with 0.1 mM CaCl2 omitted.</li>
		<li>Prepare K+ medium containing 145 mM KCl, 5 mM KOH, 10 mM HEPES, 5 mM D-glucose, and 0.1% BSA. 
		NOTE: These media were extensively optimized and are detailed in previous publications4,5.</li>
		<li>Prepare Stock ATP by weighing enough ATP powder for around 20 mL of a 100 mM stock. Dissolve the powder in 17 mL of KCl buffer (145 mM KCl, 5 mM KOH, and 10 mM HEPES, pH 7.5) and slowly, while stirring, add 2 mL of 18% (w/v) tetramethylammonium hydroxide (TMA) to the solution to bring the pH to 6.8&#8211;7.0.</li>
		<li>Adjust the final volume to 20 mL. Do not overshoot the pH past 7.0. Store the stock at -80 &#176;C; note that the ATP aliquots are stable for at least 6 months. 
		NOTE: The free molecular weight of anhydrous ATP is 551.14 g/mol, and this does not include the molecular weight of the water and disodium molecules, which may vary from batch to batch and should be taken into consideration for the calculations. TMA is toxic, and care must be taken when handling. Read the safety data sheet and prepare the stocks in a fume cabinet.</li>
		<li>Prepare stock BzATP by dissolving the BzATP in ultrapure H2O for a final stock concentration of 10 mM.</li>
	</ol>
	</li>
	<li>Create a single-cell suspension as described in steps 1.16 and 1.17. Count the cells using a hemocytometer or automatic cell counter. Resuspend the cells in the required medium (e.g., Na+ medium, culture medium) for the experiment being conducted (as described below) and place the cells on ice in the meantime.</li>
	<li>For each experiment, ensure there is enough sample to include controls for forward and side scatter, as well as for the calibration of voltage and compensation settings. Note, as an indication, that spheres grown in a T75 flask typically yield around 8 x 106 cells per flask.</li>
	<li>Set the flow cytometer settings using control samples.
	<ol>
		<li>Use forward and side scatter to selectively gate the living cells. 
		NOTE: Forward scatter provides information regarding cell size based on light diffraction, while side scatter provides a measure of internal complexity or granularity. Flow events with small forward and side scatter may be considered as dead cells.</li>
		<li>Adjust the voltage and gain of the flow cytometer according to the manufacturer&#8217;s instructions. Run a trial sample to ensure the capture of data at the maximum fluorescence intensity. No compensation is required for single channel acquisitions.</li>
	</ol>
	</li>
</ol>
3. Measuring Calcium Influx by Live-cell flow Cytometry
<ol>
	<li>Following the preparation of a single-cell suspension, resuspend the cells in 1 mL of calcium-free Na+ medium and load them with 2 ng/mL of calcium indicator dye according to the manufacturer&#8217;s protocol (refer to the Table of Materials) with 10 &#181;L of 5% pluronic acid. Incubate the cells for 30 min at 37 &#176;C.</li>
	<li>Wash the cells by adding 3&#8211;5 mL of calcium-free Na+ medium and centrifuging gently (200 x g for 4 min). Remove the supernatant and resuspend the cells in calcium-free Na+ medium, washing a second time.</li>
	<li>Resuspend the cells in 1 mL of calcium-free Na+ medium, place them on ice, and allow them to de-esterify for 30 min.</li>
	<li>Wash 1x more by adding 3&#8211;5 mL of K+ medium and centrifuging (200 x g for 4 min); then, resuspend the cells in K+ medium and aliquot them into fluorescence assisted cell sorting (FACS) tubes at a concentration of 1 x 106 cells per 500 &#181;L per FACS tube. 
	NOTE: The number of FACS tubes per sample will depend on the number of treatments and repeats that are included.</li>
	<li>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.</li>
	<li>For some samples, preincubate the cells with P2X7 receptor-specific inhibitor AZ10606120 (1 &#181;M for 10&#8211;15 min) or A438079 (10 &#181;M for 30 min) at 37 &#176;C prior to analysis.</li>
	<li>A few minutes prior to running the first sample, add CaCl2 (to a final concentration of 1 mM in the FACS tube) and place the tube in a 37 &#176;C water bath to recover.</li>
	<li>Drop a clean, small magnetic stirrer into the FACS tube and position the tube in the time module linked to a circulating 37 &#176;C 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 jacket tube adapter onto the sample platform and close the lever arm of the FACS machine.</li>
	<li>Initiate sample acquisition and run the sample for 3 min at around 1,000 events per second.</li>
	<li>At the 40 s mark, quickly remove the tube and add the P2X7 agonist, either 1 mM ATP or 300 &#181;M BzATP, and replace the tube to continue the acquisition.</li>
	<li>While the first sample is recording, prepare the second sample with CaCl2 and place it in 37 &#176;C 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, and then the acquisition of the second sample can begin as described in steps 3.8 and 3.9. Always clean the intake between samples.</li>
</ol>
4. Measuring Pore Formation by Live-cell Flow Cytometry
<ol>
	<li>Create a single-cell suspension as described in steps 1.16 and 1.17. Save a few milliliters of the old medium, use it to resuspend the cells at a concentration of 1 x 106 cells per 100 &#181;L per FACS tube, and place it on ice until ready.</li>
	<li>Prior to running the assay, add 900 &#181;L of K+ medium for a final volume of 1 mL and place the tubes in a 37 &#176;C water bath for 10 min to recover.</li>
	<li>If applicable, preincubate the cells with treatments, including the P2X7-specific inhibitors AZ10606120 (1 &#181;M for 10&#8211;15 min) or A438079 (10 &#181;M for 30 min).</li>
	<li>Immediately prior to running the assay, add 25 &#181;M ethidium bromide to the FACS tube; then, add the magnetic stirrer, place it on the FACS machine according to step 3.7, and begin the acquisition. 
	NOTE: Ethidium bromide is toxic, and care must be taken when handling it. Dispose of used FACS tubes appropriately.</li>
	<li>To induce the formation of pores in the cell membrane, add 1 mM ATP or 100 &#181;M BzATP 40 s after the start of the acquisition.</li>
	<li>Run the samples at around 1,000 events per second for 6 min.</li>
	<li>While the first sample is running, take the second sample from the ice and place it in the 37 &#176;C water bath to allow sufficient time for the cells to recover prior to the analysis. Once the first sample has finished with the acquisition, clean the intake by running a water sample; then, the second sample can be placed on the machine to begin recording as described in steps 3.8 and 3.9.</li>
</ol>
5. Measuring Phagocytosis by Live-cell Flow Cytometry
<ol>
	<li>Create a single-cell suspension as described in steps 1.16 and 1.17. Resuspend the cells in conditioned medium and aliquot them into FACS tubes at a concentration of at least 1 x 106 cells per 100 &#181;L per FACS tube. Dilute the cells to a final concentration of 1 x 106 cells/mL with Na+ medium (e.g., add 900 &#181;L of Na+ medium) and place the cells on ice until the analysis is performed.</li>
	<li>Use 1 &#181;m of YG latex beads (microspheres) as phagocytic targets for real-time phagocytosis assays. 
	NOTE: Other colors may also be substituted, but differently sized beads were found to be inadequate targets of phagocytosis4.</li>
	<li>Prior to running the first sample, transfer the cells to a 37 &#176;C water bath and incubate them for approximately 7&#8211;10 min to allow the cells to recover.</li>
	<li>Add any treatments requiring preincubation to their respective tubes, including 1 mM ATP for 15 min, 300 &#181;M oxidized ATP (oxATP) for 40 min, 20 &#181;M cytochalasin D for 20 min, and 4% paraformaldehyde (PFA) for 20 min.
	<ol>
		<li>No preincubation is required for 5% human serum. If treatments are added at approximately the same time, the samples can be run in reverse order while the others continue to incubate. For example, run the controls and serum first, then the ATP-treated sample, followed by cytochalasin D and PFA, and oxATP last.</li>
	</ol>
	</li>
	<li>Place the sample on the cytometer with the magnetic stirrer as described in steps 3.8 and 3.9 and, then, initiate sample acquisition.</li>
	<li>Remove the sample tube from the machine 15&#8211;20 s after the start of the acquisition, and add 5 &#181;L of undiluted YG beads. Return the sample FACS tube and continue the acquisition. Run the samples for 7&#8211;8 min at around 1,000 events/s.</li>
	<li>While the first sample is running, take the second sample from the ice and place it in a 37 &#176;C water bath to allow sufficient time for the cells to recover prior to analysis. Once the first sample is finished, clean the intake by running a water sample and, then, begin acquisition on the second sample as described in steps 3.8 and 3.9.</li>
</ol>
6. Data Analysis
<ol>
	<li>Export the data to a spreadsheet. The data analysis will depend on the experimental question. 
	NOTE: Be aware that different runs may have different baseline intensities, so it is important to run the assay for a designated time at the start (around 40 s) prior to adding any agonists and to normalize the data by calculating the change in fluorescence (the fluorescence at any given time point [F] divided by the fluorescence at time point zero [F0], or F/F0).</li>
	<li>To quantify the rate or kinetics of the P2X7 function in question, calculate the area under the curve or the sum of the trapezoids created under the curve for each 10 s time period16.</li>
	<li>To determine the effects of treatments, average the fluorescence intensity over the final 10&#8211;20 s of recording and compare the treatments. Determine the significance by t-test or analysis of variance.</li>
</ol>

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J., Rathsam, C., Stokes, L., McGeachie, A. B., Wiley, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+ATP+dissociates+nonmuscle+myosin+from+P2X(7)+complex:+this+dissociation+regulates+P2X(7)+pore+formation.">Extracellular ATP dissociates nonmuscle myosin from P2X(7) complex: this dissociation regulates P2X(7) pore formation.</a> American Journal of Physiology-Cell Physiology. 297 (2), C430-C439 (2009).</li><li>Gu, B. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=P2X7+receptor-mediated+scavenger+activity+of+mononuclear+phagocytes+toward+non-opsonized+particles+and+apoptotic+cells+is+inhibited+by+serum+glycoproteins+but+remains+active+in+cerebrospinal+fluid.">P2X7 receptor-mediated scavenger activity of mononuclear phagocytes toward non-opsonized particles and apoptotic cells is inhibited by serum glycoproteins but remains active in cerebrospinal fluid.</a> Journal of Biological Chemistry. 287 (21), 17318-17330 (2012).</li><li>Bhattacharya, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+Advances+in+CNS+P2X7+Physiology+and+Pharmacology:+Focus+on+Neuropsychiatric+Disorders.">Recent Advances in CNS P2X7 Physiology and Pharmacology: Focus on Neuropsychiatric Disorders.</a> Frontiers in Pharmacology. 9 (30), (2018).</li><li>Burnstock, G., Knight, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+potential+of+P2X7+receptors+as+a+therapeutic+target,+including+inflammation+and+tumour+progression.">The potential of P2X7 receptors as a therapeutic target, including inflammation and tumour progression.</a> Purinergic Signalling. 14 (1), 1-18 (2018).</li><li>Alves, L. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pore+forming+channels+as+a+drug+delivery+system+for+photodynamic+therapy+in+cancer+associated+with+nanoscintillators.">Pore forming channels as a drug delivery system for photodynamic therapy in cancer associated with nanoscintillators.</a> Oncotarget. 9 (38), 25342-25354 (2018).</li><li>Pacheco, P. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=P2X7+receptor+as+a+novel+drug+delivery+system+to+increase+the+entrance+of+hydrophilic+drugs+into+cells+during+photodynamic+therapy.">P2X7 receptor as a novel drug delivery system to increase the entrance of hydrophilic drugs into cells during photodynamic therapy.</a> Journal of Bioenergetics and Biomembranes. 48 (4), 397-411 (2016).</li></ol>

The authors have nothing to disclose.

This paper presents a detailed protocol for the analysis of P2X7 receptor function in neural progenitor cell cultures derived from the adult neurogenic niches. The potential applications for adult neural progenitor cells range from research to therapeutic purposes, and so the method of culture must be robust and reproducible. There are a number of key aspects to this protocol that may impact the quality of the endpoint culture. Once removed from the skull, the brain should not be allowed to dry and the dissection should be performed as quickly as possible. Particularly with the hippocampus, extra care taken to remove any blood vessels or membranous tissue will result in superior progenitor cell yields. The dissociation and trituration process can heavily impact the number of spheres obtained in a culture; agitating the tissue during the incubation with trypsin-EDTA will result in a more homogeneous solution. The use of a fire-polished glass pasture pipette over a P1000 plastic pipette tip is highly recommended to reduce cell death and improve the resulting culture. Avoid overtriturating. Despite these precautions, the procedure can create a lot of debris in the P0 culture, and to avoid losing progenitor cells, washing or feeding the culture should be avoided until spheres have formed.
A number of differences between the hippocampal and SVZ cultures will be obvious at P0. Hippocampal cultures yield fewer spheres, and these generally adhere. Use a pipette tip to gently lift off the spheres for the initial passage. Adherent spheres were not observed in subsequent passages. Different brands of tissue culture flasks may cause the spheres, particularly hippocampal spheres, to adhere and grow as colonies on the bottom of the dish. This was not found to alter any downstream results for this protocol but should be monitored, and consistency should be maintained where possible.
Previous methods used to measure P2X7 receptor function, such as patch clamping to record calcium influx, are time-consuming and laborious and may only provide information on a single cell. This protocol presents a rapid and reproducible method to analyze all three main functions of P2X7 receptors using one machine. Time-resolved live-cell flow cytometry allows for whole population analysis and provides the researcher with information regarding the kinetics of calcium influx, pore formation, and/or phagocytic function. In addition to this, flow cytometry can be easily used as a method for assessing marker expression patterns and population analysis based on cell size or protein expression levels.
When conducting these experiments, differences in maximal calcium influx, ethidium uptake, or phagocytosis rates may be observed between repeats. To minimize this, the sphere size, culture conditions, and feeding regime must be consistent as the health of the cells will have a significant impact on the results obtained. The time on ice can also influence the data, so ensure everything is prepared ahead of time so that the time on ice is minimal. Ensure that the calcium indicator dye loading time is consistent. Another factor that may lead to large inconsistencies in the maximal calcium recordings is the variation between ATP batches. The preparation of ATP stocks is crucial, and the use of different batches for the same experiments should be avoided. Comparing old and new batches to ensure the ATP is consistent is also recommended. The effectiveness of P2X7 antagonists may also be cell-line- and batch-dependent, so optimization of incubation times and concentrations may be required.
It is worth noting that calcium influx/efflux is one of the most fundamental and complex cellular functions and can be mediated by many receptors. The ATP-induced calcium influx, as a classic measurement for P2X7 channel/pore function, may not accurately reflect the true function of P2X7 receptors, as ATP may also activate P2Y receptors to release intracellular calcium. In this case, barium may be a better cation to use instead of calcium as its influx is unidirectional16. To differentiate the contribution from P2Y receptors in calcium influx, conditions where 1 mM EDTA or ethylene glycol-bis(&#946;-aminoethyl ether)-N,N,N&#8242;,N&#8242;-tetraacetic acid (EGTA) is added to the K+ medium instead of CaCl2 may be used in this assay.
This protocol may also be easily adapted to suit other cell types and can be useful for investigating the functionality of alternate ion channel receptors or receptors that participate in phagocytosis. This method may also be adapted to a flow cytometry machine without a time module. As an example, phagocytosis assays may be performed where YG beads are added 7&#8211;8 min prior to the analysis by conventional flow cytometry. Keep the cells at 37 &#176;C and continuously swirl them. This will not provide real-time information but differences in the mean final fluorescence will still inform the researcher regarding the functionality of P2X7 receptors.
Interest in P2X7 receptors as a drug target21,22 or even as a drug delivery route23,24 is growing rapidly, and so methods to study this enigmatic receptor must be continuously adapted and improved upon to facilitate these studies. This protocol outlines methodologies that may be used to explore P2X7 function in adult neural progenitor cells, and it is hoped that achieving a greater understanding of P2X7 receptors in the neurogenic niches may lead to advancements in the treatment of stroke and other ischemic injuries.

The study of purinergic signaling is broad and multifaceted, involving cellular physiology, biochemistry, and pharmacology. Purinergic signaling is involved in an infinite number of cellular and molecular processes, from cancer and cell cycle regulation to cell-cell communications and stem cell biology; as such, there often exists a potential to modulate purinergic signaling for a therapeutic benefit. Of the purinergic receptors, P2X7 receptor has received significant attention in recent years due to its potential as a therapeutic target for numerous inflammatory conditions1. Methods to study the receptor have evolved and been adapted over the years to facilitate this research2,3,4,5. Here, we describe a live-cell flow cytometry method to investigate the multiple functions of P2X7 receptors in adult neural progenitor cells derived from the subventricular zone (SVZ) and the hippocampal dentate gyrus.
The P2X7 receptor was first described as the P2Z receptor, or the &#8216;cell death&#8217; receptor, as its activation with high concentrations of adenosine triphosphate (ATP) results in the formation of a large transmembrane pore permeable to molecules up to 900 Da6. This leads to cytoskeletal rearrangement, transmembrane pore formation, and, potentially, apoptosis and/or necrosis7. Traditionally, this function of P2X7 is quantified by the uptake of large molecular weight dyes such as YO-PRO-1 or ethidium bromide, which fluoresce when intercalated with DNA3,8. Plate reader methods, which are rapid and allow for upscaling, generally do not allow for the observation of kinetics. The method described here is based on ethidium uptake and allows the fluorescence increase to be observed over time, providing a greater depth of information with regard to the speed of pore formation. P2X7 receptors have since been shown to facilitate a number of nonimmune functions, with distinct responses depending on exposure time and agonist concentration9,10. Brief activation by lower concentrations of ATP results in cation influx for the purposes of neurotransmitter and signal transduction11. Using flow cytometry to measure calcium influx overcomes the problems associated with cumbersome and technically challenging methods&#8212;particularly, patch clamping to measure inward currents which provide invaluable details as to the change in potential across a cell membrane but do not allow for population analysis2. The third function of P2X7 receptors occurs in the absence of ATP, where P2X7 receptors have been demonstrated to facilitate phagocytosis in both the immune system and the nervous system9,12,13. Advancements in microscopy techniques have allowed the visualization of cytoskeletal rearrangements during the uptake process, although quantification and population analysis can still present a challenge.
The live-cell flow cytometry method detailed here allows for the investigation of all three main functions of P2X7 receptors in real-time. The inclusion of a time module device on the flow cytometer allows temperature control and continual stirring of cells in suspension. Agonist and antagonist stimuli can be delivered within a second, allowing the near uninterrupted measurement of the cellular response. This presents a rapid and simple method to reproducibly quantify function while avoiding the use of multiple assay systems. It is important to note that this protocol may easily be adapted to suit any cell type and could be used to examine other receptor subtypes given the inclusion of specific agonists or inhibitors, depending on their properties.&#160;

<table><tbody><tr><td>A438079</td><td>Tocris</td><td>2972/10</td><td></td></tr><tr><td>ATP</td><td>Sigma-Aldrich</td><td>A2383</td><td></td></tr><tr><td>AZ10606120</td><td>Tocris</td><td>3323/10</td><td></td></tr><tr><td>bzATP</td><td>Sigma-Aldrich</td><td>B6396</td><td></td></tr><tr><td>Cytochalasin D</td><td>Sigma-Aldrich</td><td>C8273</td><td></td></tr><tr><td>FACSCalibur</td><td>Becton Dickinson&amp;nbsp;</td><td></td><td></td></tr><tr><td>Fluo-8AM</td><td>AAT-Bioquest</td><td>21080</td><td>Fluo-4AM and Fura-Red AM have also been used successfully&amp;nbsp;</td></tr><tr><td>Fluoresbrite YG Microspheres&amp;nbsp;</td><td>Polysciences Inc</td><td>17154-10</td><td>1.00 &amp;micro;m, yellow-green</td></tr><tr><td>Glutamine</td><td>ThermoFisher Scientific</td><td>25030081</td><td>200 mM</td></tr><tr><td>HBSS</td><td>ThermoFisher Scientific</td><td>14170112</td><td></td></tr><tr><td>Heparin</td><td>Sigma-Aldrich</td><td>H3149</td><td></td></tr><tr><td>NeuroCult Basal Medium</td><td>Stemcell Technologies</td><td>5700</td><td>Mouse and rat</td></tr><tr><td>NeuroCult Proliferation Supplement</td><td>Stemcell Technologies</td><td>5701</td><td>Mouse and rat</td></tr><tr><td>Oxidized ATP</td><td>Sigma-Aldrich</td><td>A6779</td><td></td></tr><tr><td>Pluronic&amp;nbsp;F-127</td><td>Sigma-Aldrich</td><td>P2443</td><td>pluronic acid</td></tr><tr><td>Recombinant Murine EGF</td><td>Peprotech</td><td>315-09</td><td></td></tr><tr><td>Recombinant Murine FGF-basic</td><td>Peprotech</td><td>450-33</td><td></td></tr><tr><td>Tetramethylammonium Hydroxide</td><td>Sigma-Aldrich</td><td>T7505</td><td></td></tr><tr><td>Time Zero Module</td><td>Cytek Biosciences</td><td></td><td></td></tr><tr><td>Tissue culture flasks</td><td>BD Falcon (Corning)</td><td>353108 (T25), 353136 (T75)</td><td>Blue vented screw cap</td></tr><tr><td>TrypLE Express</td><td>Gibco</td><td>12604013</td><td></td></tr><tr><td>Trypsin-EDTA (0.25%)</td><td>ThermoFisher Scientific</td><td>25200056</td><td>with phenol red</td></tr><tr><td>UltraPure Ethidium Bromide</td><td>ThermoFisher Scientific</td><td>15585011</td><td>10 mg/mL</td></tr></tbody></table>

real-time live-cell flow cytometry to investigate calcium influx, pore formation, and phagocytosis by p2x7 receptors in adult neural progenitor cells

Live-cell flow cytometry is increasingly used among cell biologists to quantify biological processes in a living cell culture. This protocol describes a method whereby live-cell flow cytometry is extended upon to analyze the multiple functions of P2X7 receptor activation in real-time. Using a time module installed on a flow cytometer, live-cell functionality can be assessed and plotted over a given time period to explore the kinetics of calcium influx, transmembrane pore formation, and phagocytosis. This simple method is advantageous as all three canonical functions of the P2X7 receptor can be assessed using one machine, and the gathered data plotted over time provides information on the entire live-cell population rather than single-cell recordings often obtained using technically challenging patch-clamp methods. Calcium influx experiments use a calcium indicator dye, while P2X7 pore formation assays rely on ethidium bromide being allowed to pass through the transmembrane pore formed upon high agonist concentrations. Yellow-green (YG) latex beads are utilized to measure phagocytosis. Specific agonists and antagonists are applied to investigate the effects of P2X7 receptor activity. Individually, these methods can be modified to provide quantitative data on any number of calcium channels and purinergic and scavenger receptors. Taken together, they highlight how the use of real-time live-cell flow cytometry is a rapid, cost-effective, reproducible, and quantifiable method to investigate P2X7 receptor function.

Neural progenitor cell cultures
Neural progenitor sphere cultures derived using this method should be phase bright and have a smooth round edge (Figure 1A,B). In healthy cultures, small microspikes can be observed on the edges (Figure 1C). At late passages, or if fed inadequately, spheres can form a hollow cup shape (Figure 1D) or large oblong shapes (Figure 1E, indicated by arrow). These cultures should not be used for flow cytometry or any other downstream applications, as these features it may be indicative of differentiation. To confirm the neural progenitor status, the cells were plated on glass coverslips coated with poly-L-ornithine and laminin for immunocytochemistry (Figure 1F and, at higher confluency, Figure 1G). Cells were stained for GFAP, nestin, Sox2, vimentin, ASCL1, BLBP, Prox1, and DCX to identify the cells as Type 2 progenitor cells (hippocampus) or Type C progenitor cells (SVZ)9. Cells should have a well-defined nucleus and extended processes.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59313/59313fig1.jpg" /> 
Figure 1: Representative hippocampal neural progenitor cell culture. (A) Hippocampal neural progenitor cells are isolated from adult mice and cultured as neurospheres until approximately 100 to 150 &#181;m in diameter. (B) Neurospheres should have a smooth periphery, (C) and small microspikes may be observed on their surface. When spheres are too long in culture, they can form (D) cup or (E) oblong shapes. These cultures should not be used for experiments. To confirm the neural progenitor status of the cells, seed them as a single-cell suspension on poly-L-ornithine (PLO) and laminin-coated glass coverslips for immunochemistry. Cells should have a small soma and branching processes, (F) at low confluency and (G) ready for immunochemistry. Scale bars = 100 &#181;m. <a href="https://www.jove.com/files/ftp_upload/59313/59313fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Calcium influx by live-cell flow cytometry
This protocol allows for the analysis of P2X7 receptor function as a calcium channel in real-time. The kinetics of receptor function, as well as the effects of different agonists and antagonists, can also be assessed. When plotted over time, calcium influx in the hippocampal and SVZ neural progenitor cells was generally similar (Figure 2A and Figure 2B, respectively). Agonists (either ATP or BzATP) were added at the 40 s mark, as indicated by the red arrow. For a brief moment, the tube is removed from the recording point to add the agonist, resulting in data points of zero. This will allow for the identification of the time when the agonist was added. 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, a slower signaling pathway which releases calcium from the endoplasmic reticulum. The inclusion of P2X7 antagonists A438079 and AZ10606120 (data not shown) reduced the calcium influx in response to agonist application.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59313/59313fig2.jpg" /> 
Figure 2: Live-cell calcium influx in neural progenitor cells from the hippocampus and SVZ. P2X7 receptor calcium channel function was demonstrated in (A) hippocampal and (B) SVZ-derived progenitor cells by changes in Fluo-8 fluorescence. Application of the general P2X agonist ATP and the P2X7 agonist BzATP result in P2X7 ion channels opening, allowing calcium influx. The influx was blocked with the P2X7-specific inhibitors A438079 or AZ10606120 (data not shown). F = fluorescence; F0 = fluorescence at time point zero. <a href="https://www.jove.com/files/ftp_upload/59313/59313fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Pore formation by live-cell flow cytometry
Transmembrane pore formation is a canonical feature of P2X7 receptors, results in macromolecule exchange, and can lead to cell death. Ethidium+ is a large molecule (314 Da) excluded from healthy cells; its uptake and subsequent intercalation with DNA results in fluorescent emissions and can be used to assess the ability of P2X7 receptors to form transmembrane pores. Following the application of the agonists ATP (1 mM ATP) and BzATP (100 &#956;M) at the 40 s (indicated by the arrow), time-resolved flow cytometry captures the ethidium bromide entering the cells in real-time (Figure 3A). This effect was attenuated by the P2X7-specific inhibitor AZ10606120. The ethidium bromide uptake assay demonstrates a functional P2X7 receptor C-terminus17 and is good evidence for full-length P2X7 receptor expression. ATP concentration-response assays illustrate the effects of agonist concentration on P2X7 pore formation, using change in ethidium bromide fluorescence over time (Figure 3B). Agonist dose concentration curves together with receptor-specific inhibitors provide strong evidence for receptor activation.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59313/59313fig3.jpg" /> 
Figure 3: P2X7 transmembrane pore formation measured by ethidium uptake. The addition of ethidium bromide moments before the start of acquisition is used to measure the formation of P2X7 transmembrane pores. High concentrations of ATP and BzATP result in (A) P2X7 receptor pore formation, allowing ethidium bromide to enter the cell. The P2X7 inhibitor AZ10606120 attenuates this phenomenon and provides evidence for functional P2X7 receptors. (B) ATP concentration-response assays demonstrated significant pore formation at 500 &#956;M and 1 mM but not at lower concentrations. <a href="https://www.jove.com/files/ftp_upload/59313/59313fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Phagocytosis by live-cell flow cytometry
Our group has previously demonstrated that extracellular ATP inhibits P2X7-mediated phagocytosis by dissociating the P2X7 C-terminus from the cytoskeleton, specifically, nonmuscle myosin IIA18,19. This method expands on these findings to demonstrate P2X7 receptor involvement in phagocytosis by hippocampal and SVZ neural progenitor cells in real-time (Figure 4, an example of hippocampal phagocytosis). Uninhibited phagocytosis (control) levels of 1 &#181;m YG latex beads were established as the positive control. ATP inhibited the phagocytosis of YG beads to the same extent as the nonspecific inhibitors, namely PFA fixation and the actin polymerization inhibitor cytochalasin D, while 5% serum abolished all innate phagocytosis20.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59313/59313fig4.jpg" /> 
Figure 4: YG bead uptake demonstrating the phagocytic capacity of neural progenitors via P2X7 receptors. YG bead uptake by neural progenitor cells is observable using live-cell flow cytometry in real-time. Control levels of phagocytosis are established initially, and if the number of cells allows, reconfirmed at the end of the run. Involvement of P2X7 receptors is indicated by the inhibition of phagocytosis in the presence of ATP, as this dissociates the C-terminus from the membrane cytoskeleton, preventing P2X7-mediated cytoskeletal rearrangements. The application of ATP blocked phagocytosis to the same extent as the use of nonspecific inhibitors of phagocytosis, including paraformaldehyde (PFA) and cytochalasin D (CytD). <a href="https://www.jove.com/files/ftp_upload/59313/59313fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Real-time Live-cell Flow Cytometry to Investigate Calcium Influx, Pore Formation, and Phagocytosis by P2X7 Receptors in Adult Neural Progenitor Cells at JoVE.

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

Providing single-cell sensitivity, real-time flow cytometry is uniquely suited to quantify multimodal receptor functions of live cultures. Using adult neural progenitor cells, the P2X7 receptor function was assessed via calcium influx detected by calcium indicator dye, transmembrane pore formation by ethidium bromide uptake, and phagocytosis using fluorescent latex beads.

Live-cell flow cytometry is increasingly used among cell biologists to quantify biological processes in a living cell culture. This protocol describes a ...

real-time-live-cell-flow-cytometry-to-investigate-calcium-influx-pore

Research

JoVE Journal

Developmental Biology

9.1K Views.  Griffith University. P2X7 receptoren zijn zeer veelzijdig en functioneren anders, afhankelijk van de hoeveelheid agonistische aanwezig. Op deze manier heeft P2X7 verschillende functies en kan de fysiologie van veel verschillende systemen worden gereguleerd. Dit protocol maakt het mogelijk om de drie belangrijkste functies van de P2X7-receptor te onderzoeken.Het is een snelle, reproduceerbare en kwantificeerbare methode om zijn functie te begrijpen. Onze focus op hoe P2X7 receptoren kunnen moduleren in neur...

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

Functional Calcium Imaging in Developing Cortical Networks

A hallmark pattern of activity in developing nervous systems is spontaneous, synchronized network activity. Synchronized activity has been observed in intact spinal cord, brainstem, retina, cortex and dissociated neuronal culture preparations. During periods of spontaneous activity, neurons depolarize to fire single or bursts of action potentials, activating many ion channels. Depolarization activates voltage-gated calcium channels on dendrites and spines that mediate calcium influx. Highly synchronized electrical activity has been measured from local neuronal networks using field electrodes. This technique enables high temporal sampling rates but lower spatial resolution due to integrated read-out of multiple neurons at one electrode. Single cell resolution of neuronal activity is possible using patch-clamp electrophysiology on single neurons to measure firing activity. However, the ability to measure from a network is limited to the number of neurons patched simultaneously, and typically is only one or two neurons. The use of calcium-dependent fluorescent indicator dyes has enabled the measurement of synchronized activity across a network of cells. This technique gives both high spatial resolution and sufficient temporal sampling to record spontaneous activity of the developing network.
A key feature of newly-forming cortical and hippocampal networks during pre- and early postnatal development is spontaneous, synchronized neuronal activity (Katz &amp; Shatz, 1996; Khaziphov &amp; Luhmann, 2006). This correlated network activity is believed to be essential for the generation of functional circuits in the developing nervous system (Spitzer, 2006). In both primate and rodent brain, early electrical and calcium network waves are observed pre- and postnatally in vivo and in vitro (Adelsberger et al., 2005; Garaschuk et al., 2000; Lamblin et al., 1999). These early activity patterns, which are known to control several developmental processes including neuronal differentiation, synaptogenesis and plasticity (Rakic &amp; Komuro, 1995; Spitzer et al., 2004) are of critical importance for the correct development and maturation of the cortical circuitry.
In this JoVE video, we demonstrate the methods used to image spontaneous activity in developing cortical networks. Calcium-sensitive indicators, such as Fura 2-AM ester diffuse across the cell membrane where intracellular esterase activity cleaves the AM esters to leave the cell-impermeant form of indicator dye. The impermeant form of indicator has carboxylic acid groups which are able to then detect and bind calcium ions intracellularly.. The fluorescence of the calcium-sensitive dye is transiently altered upon binding to calcium. Single or multi-photon imaging techniques are used to measure the change in photons being emitted from the dye, and thus indicate an alteration in intracellular calcium. Furthermore, these calcium-dependent indicators can be combined with other fluorescent markers to investigate cell types within the active network.

Spontaneous activity of developing neuronal networks can be measured using AM-ester forms of calcium-sensitive indicator dyes. Changes in intracellular calcium, indicating neuronal activation, are detected as transient changes in indicator fluorescence with one- or two-photon imaging. This protocol can be adapted for a range of developmentally-dependent neuronal networks in vitro.

A hallmark pattern of activity in developing nervous systems is spontaneous, synchronized network activity. Synchronized activity has been observed in ...

Neuroscience

Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

Spontaneous intracellular calcium activity can be observed in a variety of cell types and is proposed to play critical roles in a variety of physiological processes. In particular, appropriate regulation of calcium activity patterns during embryogenesis is necessary for many aspects of vertebrate neural development, including proper neural tube closure, synaptogenesis, and neurotransmitter phenotype specification. While the observation that calcium activity patterns can differ in both frequency and amplitude suggests a compelling mechanism by which these fluxes might transmit encoded signals to downstream effectors and regulate gene expression, existing population-level approaches have lacked the precision necessary to further explore this possibility. Furthermore, these approaches limit studies of the role of cell-cell interactions by precluding the ability to assay the state of neuronal determination in the absence of cell-cell contact. Therefore, we have established an experimental workflow that pairs time-lapse calcium imaging of dissociated neuronal explants with a fluorescence in situ hybridization assay, allowing the unambiguous correlation of calcium activity pattern with molecular phenotype on a single-cell level. We were successfully able to use this approach to distinguish and characterize specific calcium activity patterns associated with differentiating neural cells and neural progenitor cells, respectively; beyond this, however, the experimental framework described in this article could be readily adapted to investigate correlations between any time-series activity profile and expression of a gene or genes of interest.

Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

We present a two-part protocol that combines fluorescent calcium imaging with in situ hybridization, allowing the experimenter to correlate patterns of calcium activity with gene expression profiles on a single-cell level.

Spontaneous intracellular calcium activity can be observed in a variety of cell types and is proposed to play critical roles in a variety of physiological ...

Live Imaging Followed by Single Cell Tracking to Monitor Cell Biology and the Lineage Progression of Multiple Neural Populations

Understanding the mechanisms that control critical biological events of neural cell populations, such as proliferation, differentiation, or cell fate decisions, will be crucial to design therapeutic strategies for many diseases affecting the nervous system. Current methods to track cell populations rely on their final outcomes in still images and they generally fail to provide sufficient temporal resolution to identify behavioral features in single cells. Moreover, variations in cell death, behavioral heterogeneity within a cell population, dilution, spreading, or the low efficiency of the markers used to analyze cells are all important handicaps that will lead to incomplete or incorrect read-outs of the results. Conversely, performing live imaging and single cell tracking under appropriate conditions represents a powerful tool to monitor each of these events. Here, a time-lapse video-microscopy protocol, followed by post-processing, is described to track neural populations with single cell resolution, employing specific software. The methods described enable researchers to address essential questions regarding the cell biology and lineage progression of distinct neural populations.

A robust protocol to monitor neural populations by time-lapse video-microscopy followed by software-based post-processing is described. This method represents a powerful tool to identify biological events in a selected population during live imaging experiments.

Understanding the mechanisms that control critical biological events of neural cell populations, such as proliferation, differentiation, or cell fate ...

Brain Pericyte Calcium and Hemodynamic Imaging in Transgenic Mice In Vivo

Recent advances in protein biology and mouse genetics have made it possible to measure intracellular calcium fluctuations of brain cells in vivo and to correlate this with local hemodynamics. This protocol uses transgenic mice that have been prepared with a chronic cranial window and express the genetically encoded calcium indicator, RCaMP1.07, under the &#945;-smooth muscle actin promoter to specifically label mural cells, such as vascular smooth muscle cells and ensheathing pericytes. Steps are outlined on how to prepare a tail vein catheter for intravenous injection of fluorescent dyes to trace blood flow, as well as how to measure brain pericyte calcium and local blood vessel hemodynamics (diameter, red blood cell velocity, etc.) by two photon microscopy in vivo through the cranial window in ketamine/xylazine anesthetized mice. Finally, details are provided for the analysis of calcium fluctuations and blood flow movies via the image processing algorithms developed by Barrett et al. 2018, with an emphasis on how these processes can be adapted to other cellular imaging data.

Brain Pericyte Calcium and Hemodynamic Imaging in Transgenic Mice In Vivo

This protocol presents steps to acquire and analyze fluorescent calcium images from brain ensheathing pericytes and blood flow data from nearby blood vessels in anesthetized mice. These techniques are useful for studies of mural cell physiology and can be adapted to investigate calcium transients in any cell type.

Recent advances in protein biology and mouse genetics have made it possible to measure intracellular calcium fluctuations of brain cells in vivo and to ...

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial function, cell death, cell metabolism and cell migration to name but a few. Cytosolic calcium is normally maintained at low nanomolar concentrations; rather it is found in high micromolar to millimolar concentrations in the endoplasmic reticulum, mitochondrial matrix and the extracellular compartment. Upon stimulation, a transient increase in cytosolic calcium serves to signal downstream events. Detecting changes in cytosolic calcium is normally performed using a live cell imaging set up with calcium binding dyes that exhibit either an increase in fluorescence intensity or a shift in the emission wavelength upon calcium binding. However, a live cell imaging set up is not freely accessible to all researchers. Alternative detection methods have been optimized for immunological cells with flow cytometry and for non-immunological adherent cells with a fluorescence microplate reader. Here, we describe an optimized, simple method for detecting changes in epithelial cells with flow cytometry using a single wavelength calcium binding dye. Adherent renal proximal tubule epithelial cells, which are normally difficult to load with dyes, were loaded with a fluorescent cell permeable calcium binding dye in the presence of probenecid, brought into suspension and calcium signals were monitored before and after addition of thapsigargin, tunicamycin and ionomycin.

Calcium is involved in numerous physiological and pathophysiological signaling pathways. Live cell imaging requires specialized equipment and can be time consuming. A quick, simple method using a flow cytometer to determine relative changes in cytosolic calcium in adherent epithelial cells brought into suspension was optimized.

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial ...

Biology

Real-Time Fluorescent Measurement of Synaptic Functions in Models of Amyotrophic Lateral Sclerosis

Before neuronal degeneration, the cause of motor and cognitive deficits in patients with amyotrophic lateral sclerosis (ALS) and/or frontotemporal lobe dementia (FTLD) is dysfunction of communication between neurons and motor neurons and muscle. The underlying process of synaptic transmission involves membrane depolarization-dependent synaptic vesicle fusion and the release of neurotransmitters into the synapse. This process occurs through localized calcium influx into the presynaptic terminals where synaptic vesicles reside. Here, the protocol describes fluorescence-based live-imaging methodologies that reliably report depolarization-mediated synaptic vesicle exocytosis and presynaptic terminal calcium influx dynamics in cultured neurons.
Using a styryl dye that is incorporated into synaptic vesicle membranes, the synaptic vesicle release is elucidated. On the other hand, to study calcium entry, Gcamp6m is used, a genetically encoded fluorescent reporter. We employ high potassium chloride-mediated depolarization to mimic neuronal activity. To quantify synaptic vesicle exocytosis unambiguously, we measure the loss of normalized styryl dye fluorescence as a function of time. Under similar stimulation conditions, in the case of calcium influx, Gcamp6m fluorescence increases. Normalization and quantification of this fluorescence change are performed in a similar manner to the styryl dye protocol. These methods can be multiplexed with transfection-based overexpression of fluorescently tagged mutant proteins. These protocols have been extensively used to study synaptic dysfunction in models of FUS-ALS and C9ORF72-ALS, utilizing primary rodent cortical and motor neurons. These protocols easily allow for rapid screening of compounds that may improve neuronal communication. As such, these methods are valuable not only for the study of ALS but for all areas of neurodegenerative and developmental neuroscience research.

Two related methods are described to visualize subcellular events required for synaptic transmission. These protocols enable the real-time monitoring of the dynamics of presynaptic calcium influx and synaptic vesicle membrane fusion using live-cell imaging of in vitro cultured neurons.

Before neuronal degeneration, the cause of motor and cognitive deficits in patients with amyotrophic lateral sclerosis (ALS) and/or frontotemporal lobe ...

Visualizing Shifts on Neuron-Glia Circuit with the Calcium Imaging Technique

Here, we report on selective in vitro models of circuits based on glia (astrocytes, oligodendrocytes, and microglia) and/or neurons from peripheral (dorsal root ganglia) and central tissues (cortex, subventricular zone, organoid) that are dynamically studied in terms of calcium shifts. The model chosen to illustrate the results is the retina, a simple tissue with complex cellular interactions. Calcium is a universal messenger involved in most of the important cellular roles. We explain in a step-by-step protocol how retinal neuron-glial cells in culture can be prepared and evaluated, envisioning calcium shifts. In this model, we differentiate neurons from glia based on their selective response to KCl and ATP. Calcium permeable receptors and channels are selectively expressed in different compartments. To analyze calcium responses, we use ratiometric fluorescent dies such as Fura-2. This probe quantifies free Ca2+ concentration based on Ca2+-free and Ca2+-bound forms, presenting two different peaks, founded on the fluorescence intensity perceived on two wavelengths.

Cell calcium imaging is a versatile methodology to study dynamic signaling of individual cells, on mixed populations in culture or even on awakened animals, based on the expression of calcium-permeable channels/receptors that gives unique functional signatures.

Here, we report on selective in vitro models of circuits based on glia (astrocytes, oligodendrocytes, and microglia) and/or neurons from peripheral ...

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, electrical excitability and cell proliferation. The diversity and specificity of Ca2+ signaling derives from mechanisms by which Ca2+ signals are generated to act over different time and spatial scales, ranging from cell-wide oscillations and waves occurring over the periods of minutes to local transient Ca2+ microdomains (Ca2+ puffs) lasting milliseconds. Recent advances in electron multiplied CCD (EMCCD) cameras now allow for imaging of local Ca2+ signals with a 128 x 128 pixel spatial resolution at rates of &#62;500 frames sec-1 (fps). This approach is highly parallel and enables the simultaneous monitoring of hundreds of channels or puff sites in a single experiment. However, the vast amounts of data generated (ca. 1 Gb per min) render visual identification and analysis of local Ca2+ events impracticable. Here we describe and demonstrate the procedures for the acquisition, detection, and analysis of local IP3-mediated Ca2+ signals in intact mammalian cells loaded with Ca2+ indicators using both wide-field epi-fluorescence (WF) and total internal reflection fluorescence (TIRF) microscopy. Furthermore, we describe an algorithm developed within the open-source software environment Python that automates the identification and analysis of these local Ca2+ signals. The algorithm localizes sites of Ca2+ release with sub-pixel resolution; allows user review of data; and outputs time sequences of fluorescence ratio signals together with amplitude and kinetic data in an Excel-compatible table.

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Here we present techniques for imaging local IP3-mediated Ca2+ events using fluorescence microscopy in intact mammalian cells loaded with Ca2+ indicators together with an algorithm that automates identification and analysis of these events.

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, ...

High-Resolution Imaging Technique for Detecting Ca2+ Microdomains in T Cells

Imaging Initial Ca2+ Microdomains in Primary T Cells

Local, sub-second Ca2+ signals, termed Ca2+ microdomains, are highly dynamic and short-lived Ca2+ signals, which result in a global [Ca2+]i elevation and might already determine the fate of a T cell. Upon T cell receptor activation, NAADP is formed rapidly, binding to NAADP binding proteins (HN1L/JPT2, LSM12) and their respective receptors (RyR1, TPC2) sitting on intracellular Ca2+ stores, like the ER and lysosomes, and leading to subsequent release and elevation of [Ca2+]i. To capture these fast and dynamically occurring Ca2+ signals, we developed a high-resolution imaging technique using a combination of two Ca2+ indicators, Fluo-4 AM and FuraRed AM. For postprocessing, an open-source, semi-automated Ca2+ microdomain detection approach was developed based on the programming language Python. Using this workflow, we are able to reliably detect Ca2+ microdomains on a subcellular level in primary murine and human T cells in high temporal and spatial resolution fluorescence videos. This method can also be applied to other cell types, like NK cells and murine neuronal cell lines.

Author Spotlight: Unraveling the Initial Activation of the Adaptive Immune Response for Therapeutic Intervention

Here, we provide a comprehensive protocol to resolve initial local Ca2+ signals, known as Ca2+ microdomains, in primary murine and human T cells using fluorescence microscopy. This protocol serves as a valuable resource for researchers examining Ca2+ signaling pathways within immune cells and to further unravel their function.

Local, sub-second Ca2+ signals, termed Ca2+ microdomains, are highly dynamic and short-lived Ca2+ signals, which result in a global [Ca2+]i elevation and ...

Immunology and Infection

Subcellular Imaging of Neuronal Calcium Handling In Vivo

Calcium (Ca2+) imaging has been largely used to examine neuronal activity, but it is becoming increasingly clear that subcellular Ca2+ handling is a crucial component of intracellular signaling. The visualization of subcellular Ca2+ dynamics in vivo,&#160;where neurons can be studied in their native, intact circuitry, has proven technically challenging in complex nervous systems. The transparency and relatively simple nervous system of the nematode Caenorhabditis elegans enable the cell-specific expression and in vivo visualization of fluorescent tags and indicators. Among these are fluorescent indicators that have been modified for use in the cytoplasm as well as various subcellular compartments, such as the mitochondria. This protocol enables non-ratiometric Ca2+ imaging in vivo with a subcellular resolution that permits the analysis of Ca2+ dynamics down to the level of individual dendritic spines and mitochondria. Here, two available genetically encoded indicators with different Ca2+ affinities are used to demonstrate the use of this protocol for measuring relative Ca2+ levels within the cytoplasm or mitochondrial matrix in a single pair of excitatory interneurons (AVA). Together with the genetic manipulations and longitudinal observations possible in C. elegans, this imaging protocol may be useful for answering questions regarding how Ca2+ handling regulates neuronal function and plasticity.

Subcellular Imaging of Neuronal Calcium Handling In Vivo

The current methods describe a non-ratiometric approach for high-resolution, sub-compartmental calcium imaging in vivo in Caenorhabditis elegans using readily available genetically encoded calcium indicators.

Calcium (Ca2+) imaging has been largely used to examine neuronal activity, but it is becoming increasingly clear that subcellular Ca2+ handling is a ...

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

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