We thank the UW-Madison flow cytometry core (P30 CA014520 and 1S10RR025483-01), and members of the Moore lab and UW-Madison community for their input. We thank our funding sources: NIH T32 T32GM008688 (to C.S.M.), Diana Jacobs Kalman Fellowship from AFAR (to C.S.M.), Wisconsin Graduate Fellowship (to C.S.M.), DP2 NIH New Innovator Award (1DP2OD025783, to D.L.M.), Vallee Scholar Award (to D.L.M.), NIH 1R56NS130450 (to D.L.M and M.C.S.), R01 CA185747 (to M.C.S.), R01 CA205101 (to M.C.S.), R01 CA211082 (to M.C.S.), and the National Science Foundation Grant No. CBET-1642287 (to M.C.S.).

All procedures in this protocol are approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Wisconsin-Madison.
1. Using a confocal microscope to image PAF in qNSCs and aNSCs to identify NSC cell-state
<ol>
	<li>Generate qNSCs and aNSCs in vitro from primary NSC cultures.
	<ol>
		<li>Culture NSCs purified from adult mouse brains in a proliferative medium for expansion (Table 1)18,<su...

<ol>
	<li>Goncalves, J. T., Schafer, S. T., Gage, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+neurogenesis+in+the+hippocampus:+From+stem+cells+to+behavior.">Adult neurogenesis in the hippocampus: From stem cells to behavior.</a> Cell. 167 (4), 897-914 (2016).</li><li>Kuhn, H. G., Dickinson-Anson, H., Gage, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurogenesis+in+the+dentate+gyrus+of+the+adult+rat:+age-related+decrease+of+neuronal+progenitor+proliferation.">Neurogenesis in the dentate gyrus of the adult rat: age-related decrease of neuronal progenitor proliferation.</a> J Neurosci. 16 (6), 2027-2033 (1996).</li><li>Silva-Vargas, V., Maldonado-Soto, A. R., Mizrak, D., Codega, P., Doetsch, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-dependent+niche+signals+from+the+choroid+plexus+regulate+adult+neural+stem+cells.">Age-dependent niche signals from the choroid plexus regulate adult neural stem cells.</a> Cell Stem Cell. 19 (5), 643-652 (2016).</li><li>Urban, N., Blomfield, I. M., Guillemot, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quiescence+of+adult+mammalian+neural+stem+cells:+A+highly+regulated+rest.">Quiescence of adult mammalian neural stem cells: A highly regulated rest.</a> Neuron. 104 (5), 834-848 (2019).</li><li>Urban, N., 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=Return+to+quiescence+of+mouse+neural+stem+cells+by+degradation+of+a+proactivation+protein.">Return to quiescence of mouse neural stem cells by degradation of a proactivation protein.</a> Science. 353 (6296), 292-295 (2016).</li><li>Kalamakis, G., 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=Quiescence+modulates+stem+cell+maintenance+and+regenerative+capacity+in+the+aging+brain.">Quiescence modulates stem cell maintenance and regenerative capacity in the aging brain.</a> Cell. 176 (6), 1407-1419 (2019).</li><li>Walsh, A. 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=Classification+of+T-cell+activation+via+autofluorescence+lifetime+imaging.">Classification of T-cell activation via autofluorescence lifetime imaging.</a> Nat Biomed Eng. 5 (1), 77-88 (2021).</li><li>Sagar, M. A. K., 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=Microglia+activation+visualization+via+fluorescence+lifetime+imaging+microscopy+of+intrinsically+fluorescent+metabolic+cofactors.">Microglia activation visualization via fluorescence lifetime imaging microscopy of intrinsically fluorescent metabolic cofactors.</a> Neurophotonics. 7 (3), 035003 (2020).</li><li>Knobloch, M., 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=Metabolic+control+of+adult+neural+stem+cell+activity+by+Fasn-dependent+lipogenesis.">Metabolic control of adult neural stem cell activity by Fasn-dependent lipogenesis.</a> Nature. 493 (7431), 226-230 (2013).</li><li>Knobloch, M., 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=A+fatty+acid+oxidation-dependent+metabolic+shift+regulates+adult+neural+stem+cell+activity.">A fatty acid oxidation-dependent metabolic shift regulates adult neural stem cell activity.</a> Cell Rep. 20 (9), 2144-2155 (2017).</li><li>Knobloch, M., 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=SPOT14-positive+neural+stem/progenitor+cells+in+the+hippocampus+respond+dynamically+to+neurogenic+regulators.">SPOT14-positive neural stem/progenitor cells in the hippocampus respond dynamically to neurogenic regulators.</a> Stem Cell Reports. 3 (5), 735-742 (2014).</li><li>Shin, 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=Single-cell+RNA-Seq+with+waterfall+reveals+molecular+cascades+underlying+adult+neurogenesis.">Single-cell RNA-Seq with waterfall reveals molecular cascades underlying adult neurogenesis.</a> Cell Stem Cell. 17 (3), 360-372 (2015).</li><li>Leeman, D. S., 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=Lysosome+activation+clears+aggregates+and+enhances+quiescent+neural+stem+cell+activation+during+aging.">Lysosome activation clears aggregates and enhances quiescent neural stem cell activation during aging.</a> Science. 359 (6381), 1277-1283 (2018).</li><li>Beckervordersandforth, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+metabolism-mediated+regulation+of+adult+neurogenesis.">Mitochondrial metabolism-mediated regulation of adult neurogenesis.</a> Brain Plast. 3 (1), 73-87 (2017).</li><li>Morrow, C. S., 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=Autofluorescence+is+a+biomarker+of+neural+stem+cell+activation+state.">Autofluorescence is a biomarker of neural stem cell activation state.</a> Cell Stem Cell. , (2024).</li><li>Kolenc, O. I., Quinn, K. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+cell+metabolism+through+autofluorescence+imaging+of+NAD(P)H+and+FAD.">Evaluating cell metabolism through autofluorescence imaging of NAD(P)H and FAD.</a> Antioxid Redox Signal. 30 (6), 875-889 (2019).</li><li>Mira, H., 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=Signaling+through+BMPR-IA+regulates+quiescence+and+long-term+activity+of+neural+stem+cells+in+the+adult+hippocampus.">Signaling through BMPR-IA regulates quiescence and long-term activity of neural stem cells in the adult hippocampus.</a> Cell Stem Cell. 7 (1), 78-89 (2010).</li><li>Morrow, C. S., 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=Vimentin+coordinates+protein+turnover+at+the+aggresome+during+neural+stem+cell+quiescence+exit.">Vimentin coordinates protein turnover at the aggresome during neural stem cell quiescence exit.</a> Cell Stem Cell. 26 (4), 558-568 (2020).</li><li>Martynoga, B., 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=Epigenomic+enhancer+annotation+reveals+a+key+role+for+NFIX+in+neural+stem+cell+quiescence.">Epigenomic enhancer annotation reveals a key role for NFIX in neural stem cell quiescence.</a> Genes Dev. 27 (16), 1769-1786 (2013).</li><li>Bin Imtiaz, M. K., Jessberger, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+adult+mouse+hippocampal+neural+stem+cells+for+fluorescence+loss+in+photobleaching+assays.">Isolation of adult mouse hippocampal neural stem cells for fluorescence loss in photobleaching assays.</a> STAR Protoc. 2 (3), 100695 (2021).</li><li>Guo, W., Patzlaff, N. E., Jobe, E. M., Zhao, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+multipotent+neural+stem+or+progenitor+cells+from+both+the+dentate+gyrus+and+subventricular+zone+of+a+single+adult+mouse.">Isolation of multipotent neural stem or progenitor cells from both the dentate gyrus and subventricular zone of a single adult mouse.</a> Nat Protoc. 7 (11), 2005-2012 (2012).</li><li>Lakowicz, J. R., Szmacinski, H., Nowaczyk, K., Johnson, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+lifetime+imaging+of+free+and+protein-bound+NADH.">Fluorescence lifetime imaging of free and protein-bound NADH.</a> Proc Natl Acad Sci U S A. 89 (4), 1271-1275 (1992).</li><li>Blacker, T. S., Duchen, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigating+mitochondrial+redox+state+using+NADH+and+NADPH+autofluorescence.">Investigating mitochondrial redox state using NADH and NADPH autofluorescence.</a> Free Radic Biol Med. 100, 53-65 (2016).</li><li>Datta, R., Heaster, T. M., Sharick, J. T., Gillette, A. A., Skala, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+lifetime+imaging+microscopy:+fundamentals+and+advances+in+instrumentation,+analysis,+and+applications.">Fluorescence lifetime imaging microscopy: fundamentals and advances in instrumentation, analysis, and applications.</a> J Biomed Opt. 25 (7), 1-43 (2020).</li><li>. ImageJ Available from: <a target="_blank" href="https://imagej.nih.gov/ij/">https://imagej.nih.gov/ij/</a> (2021)</li><li>. Becker &amp; Hichl Handbooks Available from: <a target="_blank" href="https://imagej.nih.gov/ij/">https://imagej.nih.gov/ij/</a> (2024)</li><li>. GitHub Materials Available from: <a target="_blank" href="https://github.com/chrismorrow5/Neural-Stem-Cell-Autofluorescence-Analysis/">https://github.com/chrismorrow5/Neural-Stem-Cell-Autofluorescence-Analysis/</a> (2024)</li><li>Codega, P., 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=Prospective+identification+and+purification+of+quiescent+adult+neural+stem+cells+from+their+in+vivo+niche.">Prospective identification and purification of quiescent adult neural stem cells from their in vivo niche.</a> Neuron. 82 (3), 545-559 (2014).</li><li>Llorens-Bobadilla, E., 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=Single-cell+transcriptomics+reveals+a+population+of+dormant+neural+stem+cells+that+become+activated+upon+brain+injury.">Single-cell transcriptomics reveals a population of dormant neural stem cells that become activated upon brain injury.</a> Cell Stem Cell. 17 (3), 329-340 (2015).</li></ol>

The authors declare no competing interests.

This protocol describes a live-cell, label-free, non-destructive, single-cell resolution technique that allows for the classification of NSC cell-state in vitro through imaging of autofluorescent signals in NSCs. This approach detects metabolic shifts that occur during NSC quiescence exit, which influence the optical properties of metabolic cofactors, such as NAD(P)H, and offers many advantages over existing technologies to study NSC quiescence. For example, many conventional techniques for studying qNSCs and aN...

NSCs create newborn neurons throughout life in many organisms in a process referred to as adult neurogenesis1,2. To produce newborn neurons, a qNSC first must activate, entering the cell cycle to&#160;expand the population&#160;and produce neural progenitors3,4,5,6. Although there is much known about NSC quiescence, our ability to fully identify the drivers and regulators of NSC quiescence is constrained by technical limitations that exist to isolate and identify qNSCs and their transition to activation. Autofluorescence imaging has previously been successful in studying changes in cell state in many different cell types, such as microglia and T-cells, by resolving metabolic remodeling, which influences the optical properties of autofluorescent metabolic cofactors such as nicotinamide adenine dinucleotide phosphate (NAD(P)H) and flavin adenine dinucleotide (FAD)7,8. NSCs substantially remodel their metabolic networks as they undergo quiescence exit9,10,11,12,13,14. Thus, to take advantage of these differences, NSC autofluorescence was recently used to identify and enrich the NSC activation state by detecting shifts in autofluorescence attributed to the metabolic remodeling that occurs as NSCs exit quiescence15. Imaging autofluorescence provides several technical advantages: i) it does not require the addition of exogenous labels, which can impact cell behavior; ii) it can provide high-resolution single-cell data on the NSC activation state; and iii) it does not require the destruction of the cell7,16. This protocol outlines three strategies for harnessing NSC autofluorescence to study NSC quiescent and activated cell states15.
Recently, NSCs isolated from 6-week-old male mice from the subgranular zone of the hippocampus, cultured and reversibly put into quiescence in vitro10,13,17,18,19,20,21, were found to exhibit increased levels of punctate autofluorescence (PAF) that excite between 400-600 nm and emit between 500-700 nm. This signal was specific to qNSCs compared to activated, cycling NSCs15. The ability to visually separate these two populations without the use of additional antibody markers or reporters is useful for many experimental questions on the nature of qNSCs and quiescence exits. Thus, first, this protocol describes strategies to image the PAF in qNSCs using a confocal microscope, which can be used to identify&#160;NSC activation state. Second, this protocol describes strategies to detect the PAF using fluorescence-activated cell sorting (FACS) and further describes how to sort based on this signal to enrich qNSCs or aNSCs. These strategies provide one measure that can be used to cluster and separate NSCs based on cell state.
To develop a higher resolution method of separating NSCs not only in distinct states but also as they transition through quiescence exit towards full activation, fluorescence lifetime imaging (FLIM) was performed using a multiphoton microscope to image NAD(P)H (termed Channel 1) autofluorescence and green autofluorescence (termed Channel 2; which detects both FAD autofluorescence and PAF in qNSCs) lifetimes together with their intensity. This approach capitalizes on the fact that the optical properties of molecules in the cell are dependent on their physical properties16,22. For example, NAD(P) (NAD and NADP are optically indistinguishable, and thus NAD(P) is used to refer to both species) is not autofluorescent in the oxidized state but is autofluorescent in its reduced state (NAD(P)H)23. Further, additional physical properties of autofluorescent molecules, such as their binding status to enzymes, can be extrapolated by performing fluorescence lifetime imaging7,22,24. For example, NAD(P)H has a shorter fluorescence lifetime when not bound to an enzyme22. As autofluorescent molecules such as NAD(P)H, which is involved in hundreds of metabolic reactions, are used differently by cells progressing through different states or cell behaviors, these shifts can be detected and quantified using a multiphoton microscope detecting autofluorescence lifetime23. Together with the abundance, or intensity, of the autofluorescence, these measures provide multi-dimensional information to separate NSCs into one cell state or the other and through the dynamic transitions between states. Third, this protocol describes performing, analyzing, and interpreting FLIM and intensity measures of Channel 1 (NAD(P)H) and Channel 2 (PAF) signals using a multiphoton microscope. In summary, this protocol describes a live-cell, label-free toolkit for studying NSC quiescence that provides high-resolution single-cell data on&#160;NSC state.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>40x Water objective lens</td>
			<td>Nikon</td>
			<td>MRD77410</td>
			<td>Objective lens used in multiphoton microscope in Part 3</td>
		</tr>
		<tr>
			<td>8 well cuvette</td>
			<td>Ibidi</td>
			<td>80826-90</td>
			<td>For imaging aNSCs/qNSCs</td>
		</tr>
		<tr>
			<td>Analog power meter</td>
			<td>Thorlabs</td>
			<td>PM100A</td>
			<td>Used in multiphoton microscope in Part 3</td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic (100X) (PSF)</td>
			<td>Thermo Fisher</td>
			<td>15240062</td>
			<td>Antibiotic for NSC media</td>
		</tr>
		<tr>
			<td>B-27</td>
			<td>Invitrogen</td>
			<td>17504044</td>
			<td>Nutrient supplement for NSC media</td>
		</tr>
		<tr>
			<td>BMP4</td>
			<td>Fisher Scientific</td>
			<td>5020BP010</td>
			<td>Factor for inducing quiescence</td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma</td>
			<td>A4919-25G</td>
			<td>For making BMP4</td>
		</tr>
		<tr>
			<td>Chameleon ultrafast laser</td>
			<td>Coherent</td>
			<td>N/A</td>
			<td>Laser used in multiphoton microscope in Part 3</td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Nikon</td>
			<td>C2</td>
			<td>Microscope used for Part 1</td>
		</tr>
		<tr>
			<td>DMEM/F-12 (without GlutaMAX)</td>
			<td>Invitrogen</td>
			<td>11320033</td>
			<td>Base media for NSCs</td>
		</tr>
		<tr>
			<td>DNAse</td>
			<td>Sigma</td>
			<td>D5025-15KU</td>
			<td>Added to trypsin inhibitor</td>
		</tr>
		<tr>
			<td>EdU assay kit</td>
			<td>Invitrogen</td>
			<td>C10337</td>
			<td>Proliferation assay for cell culture</td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>PeproTech</td>
			<td>AF-100-15-500UG</td>
			<td>Growth factor for NSC media</td>
		</tr>
		<tr>
			<td>FGF</td>
			<td>PeproTech</td>
			<td>100-18B</td>
			<td>Growth factor for NSC media</td>
		</tr>
		<tr>
			<td>Fluorescent activated cell sorter</td>
			<td>BD</td>
			<td>FACSAria</td>
			<td>Fluorescent Activated Cell Sorter used for Part 2</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sigma</td>
			<td>H3149-50KU</td>
			<td>Additive for NSC media</td>
		</tr>
		<tr>
			<td>L-15</td>
			<td>Invitrogen</td>
			<td>21083027</td>
			<td>For preparing trypsin inhibitor solution</td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Sigma</td>
			<td>L2020-1MG</td>
			<td>For coating glassware</td>
		</tr>
		<tr>
			<td>Nikon TiE inverted microscope</td>
			<td>Nikon</td>
			<td>N/A</td>
			<td>Microscope frame Used for Part 3</td>
		</tr>
		<tr>
			<td>PLO</td>
			<td>Sigma</td>
			<td>P3655-100MG</td>
			<td>For coating glassware</td>
		</tr>
		<tr>
			<td>SPC-150 Single photon counting electronics</td>
			<td>Becker and Hickl</td>
			<td>N/A</td>
			<td>Used in multiphoton microscope in Part 3</td>
		</tr>
		<tr>
			<td>Trypsin (for trypsinizing pellets of aNSCs that were growing as spheres or monolayers)</td>
			<td>Gibco</td>
			<td>15090046</td>
			<td>For trypsinizing neurospheres or adherent aNSCs</td>
		</tr>
		<tr>
			<td>Trypsin (for trypsinizing qNSCs)</td>
			<td>Gibco</td>
			<td>25200072</td>
			<td>For trypsinizing adherent qNSCs</td>
		</tr>
		<tr>
			<td>Trypsin inhibitor</td>
			<td>Sigma</td>
			<td>T6522-100MG</td>
			<td>For inhibiting trypsinization of aNSCs</td>
		</tr>
		<tr>
			<td>Urea crystals</td>
			<td>Sigma</td>
			<td>U5128-5G</td>
			<td>Used to collect an IRF</td>
		</tr>
		<tr>
			<td>Versene</td>
			<td>Thermo Fisher</td>
			<td>15040066</td>
			<td>For preparing trypsin</td>
		</tr>
	</tbody>
</table>

classification of neural stem cell activation state in vitro using autofluorescence

Neural stem cells (NSCs) divide and produce newborn neurons in the adult brain through a process called adult neurogenesis. Adult NSCs are primarily quiescent, a reversible cell state where they have exited the cell cycle (G0) yet remain responsive to the environment. In the first step of adult neurogenesis, quiescent NSCs (qNSCs) receive a signal and activate, exiting quiescence and re-entering the cell cycle. Thus, understanding the regulators of NSC quiescence and quiescence exit is critical for future strategies targeting adult neurogenesis. However, our understanding of NSC quiescence is limited by technical constraints in identifying quiescent NSCs (qNSCs) and activated NSCs (aNSCs). This protocol describes a new approach to identify and enrich qNSCs and aNSCs generated in in vitro cultures by imaging NSC autofluorescence. First, this protocol describes how to use a confocal microscope to identify autofluorescent markers of qNSCs and aNSCs to classify NSC activation state using autofluorescence intensity. Second, this protocol describes how to use a fluorescent activated cell sorter (FACS) to classify NSC activation state and enrich samples for qNSCs or aNSCs using autofluorescence intensity. Third, this protocol describes how to use a multiphoton microscope to perform fluorescence lifetime imaging (FLIM) at single-cell resolution, classify NSC activation state, and track the dynamics of quiescent exit using both autofluorescence intensities&#160;and fluorescence lifetimes. Thus, this protocol provides a live-cell, label-free, single-cell resolution toolkit for studying NSC quiescence and quiescence exit.

Confocal autofluorescence imaging to separate NSC cell state (Figure 1) 
To use confocal microscopy to resolve the NSC activation state, qNSCs, and aNSCs were generated in vitro using either an activation medium or quiescence medium, as described previously10,13,17,18. To detect PAF in NSCs, live qNSCs and aNSCs were imaged using the ...

Read this Scientific Article Protocol about Classification of Neural Stem Cell Activation State In Vitro using Autofluorescence at JoVE.com

Classification of Neural Stem Cell Activation State In Vitro using Autofluorescence

This protocol describes strategies to identify and enrich for cell-state in primary adult mouse neural stem cell cultures by autofluorescence imaging using i) a confocal microscope, ii) a fluorescent activated cell sorter to perform intensity imaging, or iii) a multiphoton microscope to perform fluorescence lifetime imaging.

Classification of Neural Stem Cell Activation State In Vitro Using Autofluorescence

Neural stem cells (NSCs) divide and produce newborn neurons in the adult brain through a process called adult neurogenesis. Adult NSCs are primarily ...

This protocol provides a new approach to classify neural stem cell activation state with a cohort of technical advantages. Using this protocol, we can achieve live cell label-free single cell resolution analysis of neural stem cell activation state. This protocol can be used to shed light into mechanisms regulating adult neurogenesis.Autofluorescence is much dimmer than many fluorophores researchers are used to working with. Be confident in using higher laser powers to collect your data. Demonstrating the procedure will be Chris Morrow, a former graduate student from my laboratory.Begin by configuring the confocal microscope for autofluorescence imaging. Click 405 Laser and type in the emission window under the 405 laser menu. Proceed to click TD to collect a transmitted light image and set the HV value to visualize the sample.Adjust the laser power to 3.5%and set the gain to 75. Set the zoom to two. For the confocal scanning parameters, set the pixel dwell to 0.5 and the resolution to 2048 by 2048 pixels.Using 60X oil immersion objective lens under brightfield, bring the cells into focus. Click Eye Port to switch to confocal scanning mode. Ensure that the light path is directed towards the scan head and confirm that no filter cube is obstructing the light path.Click Scan, then focus on the image and click Capture to acquire images of autofluorescence in quiescent neural stem cells and active neural stem cells. Ensure to collect single plane images with the cytoplasm of cells in focus for a representative cross-section of each cell. Start imaging with low laser powers and gradually increase the power until the autofluorescence signal is detectable without causing saturation.Analyze quiescent neural stem cells and active neural stem cells with a flow cytometer using a 130 micrometer nozzle. Apply optical conditions as per the instrument's capabilities and detect punctate autofluorescence. Collect at least 10, 000 singlet qNSCs and aNSCs in a data file.Utilize this data to design gates for collecting an enriched population of either qNSCs or aNSCs. Then sort the singlet cells into PLO laminin-coated wells filled with aNSC medium. After FACS, allow neural stem cells to incubate for three hours.To fix the cells, add 4%paraformaldehyde, ensuring it is sufficient to cover the bottom of the dish and incubate. After 15 minutes, permeabilize the cells with 0.25%Triton in PBS for 15 minutes at room temperature. Following this, treat the cells with an EdU staining solution at room temperature for 30 minutes.Next, wash the samples three times for 10 minutes each with PBS. Begin by using the oculars to locate a suitable field of view and adjust the light path to the sample on the microscope to enable imaging in the dark with the multi-photon. Set up for the collection of channel one image.Navigate to the power gain tab and adjust the gain on the PMT to 800. Click on the 2P Laser tab and select 750 nanometers for the laser wavelength and ensure the correct filter cube is used. To collect channel one image, click the Power Gain tab and set the Pockels to 30.Then proceed to the scanning section of the screen and click Live Scan to start preview mode. Incrementally increase the Pockels to achieve a suitable field of view at low laser power. Once a suitable field of view has been identified, adjust the laser power to 3.6 milliwatts.Then proceed to the scanning section and click single scan to capture a channel one image over 60 seconds. To collect a channel two image, click on the 2P Laser tab and select 890 nanometers for the laser. Replace the filter cube with the channel two emission cube.Initiate the preview mode. Increase the Pockels until the power meter shows approximately seven milliwatts and the CFD counts are between 10, 000 and 100, 000. Click on single scan and capture a channel two image over a duration of 60 seconds.Confocal autofluorescence imaging was employed to differentiate between quiescent neural stem cells and activated neural stem cells. It was found that qNSCs display a higher number of PAF than aNSCs, demonstrating the use of autofluorescence as a marker for cell state identification. Using FACS, NSC cell states were enriched based on autofluorescence.Cells sorted from the high autofluorescence gate showed a lower proliferation rate compared to the mixed sample, while those from the low autofluorescence gate were more proliferative, confirming the enrichment of NSC activation states. Further, FLIM was utilized to assess NSC autofluorescence and discern the activation state of NSCs. Notably, qNSCs showed a higher mean lifetime of fluorescence in channel one, but a lower proportion of the fluorescence component alpha one compared to aNSCs.A logistic regression model incorporating data from both channels yielded a near perfect predictive capacity for NSC activation states as indicated by a receiver operator characteristic curve underscoring the potential of FLIM in classifying NSC states. Following this procedure, it would be good validate results using other approaches to measure neural stem cell activation state.

classification-neural-stem-cell-activation-state-vitro-using

Research

JoVE Journal

Neuroscience

451 visualizações.  University of Wisconsin-Madison. Este protocolo fornece uma nova abordagem para classificar o estado de ativação de células-tronco neurais com uma coorte de vantagens técnicas. Usando este protocolo, podemos obter uma análise de resolução de célula única sem marcação de células vivas do estado de ativação de células-tronco neurais. Este protocolo pode ser usado para lançar luz sobre os mecanismos que regulam a neurogênese adulta.A autofluorescência é muito mais frac...