We acknowledge the technical and theoretical contributions in spectral cytometry of K. Futamura, who also critically revised the manuscript. We are also indebted to C. Ait-Mansour, P.-H. Commere, A. Bandeira, and P. Pereira for critically reading the manuscript and for endless technical advice and support. We also thank P. Pereira for the gift of the anti V&#947;7-APC and V&#948;4-Biotin labeled antibodies. &#160;We aknowledge the Centre d&#8217;Enseignements &#160;from Pasteur Institute for welcoming and supporting the filming logistics. This work was supported by the Pasteur Institute, Institut National de la Sant&#233; et de la Recherche M&#233;dicale (INSERM); the Swiss National Science Foundation and Pasteur Bourse Roux (S.S.); Funda&#231;&#227;o para a Ci&#234;ncia e a Tecnologia - SFRH/BD/74218/2010 (M.V.); and Universit&#233; Paris Diderot, the Agence Nationale de la Recherche (ANR) project Twothyme, the ANR, and Program REVIVE (Investment for the Future) (A.C.).

All experiments were performed according to the Pasteur Institute Ethic Charter and the EU guidelines and were approved by the French Agriculture Ministry.
1. Cell Suspension Preparation from Adult Mouse Organs
<ol>
	<li>Isolation of splenocytes
	<ol>
		<li>Euthanize adult mice by cervical dislocation. Make an incision at the abdomen midline and open the skin with scissors. Harvest the spleen with forceps.</li>
		<li>Crush the spleen between 2 glass microscope slides to dissociate the cells and dilute them in 5 mL of Hank&#39;s Balanced Salted Solution (HBSS) containing 1% fetal calf serum (FCS).</li>
		<li>Centrifuge for 10 min at 120 x g and 4 &#176;C. Discard the supernatant.</li>
		<li>Resuspend the pellet in 5 mL of HBSS 1% FCS. Count the cells with a Neubauer chamber using a Trypan blue vital stain. 
		NOTE: 80 million cells should be obtained from one adult spleen.</li>
	</ol>
	</li>
	<li>Isolation of cells from the small intestine
	<ol>
		<li>Euthanize adult mice by cervical dislocation. Using scissors, make an incision in the skin at the abdomen midline. Grasp the small intestine with forceps in one hand and use scissors to cut it at both ends: first, right after the stomach and then at the end of the small intestine, just before the large intestine. Flush the small intestine with 1x Dulbecco&#39;s Phosphate Buffered Saline (DPBS) using a 2 mL syringe with a needle.</li>
		<li>Put the small intestine on a paper towel and carefully remove the Peyer&#39;s patches with a pair of sharp scissors.</li>
		<li>Use scissors to open the intestine on its long side by inserting one branch of the scissors inside the intestine. Cut the opened intestine into 1-cm pieces using sharp scissors.</li>
		<li>Incubate for 30 min at 37 &#176;C under constant stirring in 30 mL of HBSS with 10% FCS.</li>
		<li>Place the cell suspension with the remaining non-dissociated fragments in a 50 mL plastic tube and vortex for 5 min at high speed. NOTE: There is no further dissociation of the tissue.</li>
		<li>Incubate for 10 min on ice to allow the large, non-dissociated fragments to pellet.</li>
		<li>Collect the supernatant and concentrate it by centrifugation for 7 min at 120 x g.</li>
		<li>Discard the supernatant and resuspend the pellet in 10 mL of HBSS with 1% FCS. Count cells with a Neubauer chamber using a Trypan blue vital stain. 
		NOTE: ~60 million intra-epithelial cells (IELs) should be obtained from one adult small intestine.</li>
	</ol>
	</li>
	<li>Panel design strategy
	<ol>
		<li>Prepare the panels with antibodies directly coupled with fluorochromes. 
		NOTE: All antibodies are previously titrated to obtain the optimal definition of the cell populations (the titration can vary with the antibody batch). The antibodies used in the 19-color panel to stain splenocytes are listed in Table 1.</li>
		<li>Construct multi-color panels for cytometry. As this can be a challenging task, use the following guidelines to optimize the panels:
		<ol>
			<li>Check the spectral configuration and the fluorescent dyes that can be used on the instrument.</li>
			<li>Use the Brightness Index/Staining Index information provided by the manufacturer. 
			NOTE: The Brightness Index/Staining Index is the relative indicator of fluorescence intensity versus background for each fluorochrome.</li>
			<li>Choose the antibodies following the rules below:
			<ol>
				<li>Choose the brightest fluorochromes for proteins that are weakly expressed and the dimmest fluorochromes for more highly expressed proteins. 
				NOTE: For example, CD45, CD4, and CD8 are highly expressed and can be used with dim fluorochromes.</li>
				<li>Start designing the panel by first choosing the antibodies with the fewest fluorescent dye possibilities.</li>
				<li>If possible, choose fluorescent dyes with the smallest overlap of emission spectra for markers expressed on the same cell types. 
				NOTE: Tandems (e.g., PE-Cy5, PE-Cy7, and PerCP-Cy5.5) should be used with caution, as they are more susceptible to degradation by light exposure.</li>
			</ol>
			</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Staining of cells for cytometry analysis
	<ol>
		<li>Prepare one 5 mL tube with 1 x 106 splenocytes and one 5 mL tube with 1 x 106 intestinal cells and keep them unstained for use as negative controls.</li>
		<li>Prepare and label one 5 mL tube per sample and per panel according to Table 1. Prepare the mix of antibodies, according to Table 1, in HBSS 1% FCS.</li>
		<li>Transfer 1 x 106 cells &#8211; either splenocytes or intestinal cells &#8211; into each 5 mL tube. Add 2 mL of HBSS 1% FCS and centrifuge for 5 min at 4 &#176;C and 120 x g. Discard the supernatant and resuspend the pellet in 50 &#181;L of antibody mix.</li>
		<li>Label the splenocytes in 2 steps. First, use 50 &#181;L of the mix containing the antibodies labeled with PE-Cyanine 5, PerCP-Cyanine 5.5, and PerCP (with Fc receptor blocking) in a 5 mL tube. After 20 min of incubation in the dark at 4 &#176;C, add 50 &#181;L of the mix containing all the other antibodies to a 5 mL tube. 
		NOTE: This strategy limits steric hindrance.</li>
		<li>Incubate for 20 min in the dark at 4 &#176;C.</li>
		<li>Add 2 mL of HBSS 1% FCS and centrifuge for 5 min at 4 &#176;C and 120 x g. Discard the supernatant and resuspend the pellet in 200 &#181;L of HBSS 1% FCS with 0.5 &#181;g/mL propidium iodide (PI).</li>
	</ol>
	</li>
</ol>
2. Preparation of Single-staining for Each Fluorochrome on Commercial Compensation Bead Microspheres (e.g., UltraComp eBeads), Hereafter Referred to as Beads
<ol>
	<li>Prepare and label as many 5 mL tubes as the number of antibodies that are used in the panels. Place 1 drop of beads in each tube and add 1 &#181;L of antibody per tube. 
	NOTE: The beads used here (see the table of materials) contain stained (positive) and unstained (negative) beads. Antibodies are put in excess on the beads to ensure a bright signal and thus a clear spectral signature for each dye. This is required by the software to be able to make a precise unmixing.</li>
	<li>Incubate for 20 min in the dark at 4 &#176;C.</li>
	<li>Add 2 mL of HBSS 1% FCS and centrifuge for 5 min at 120 x g. Discard the supernatant and resuspend the pellet in 100 &#181;L of HBSS 1% FCS.</li>
</ol>
3. Cell Suspension Preparation from Embryonic Mouse Hearts
<ol>
	<li>Dissections of embryos
	<ol>
		<li>Plan to obtain timed-pregnant females in advance by mating 2 females with one male (always in the male&#39;s cage) at the end of the day (between 17 and 19 h). The next morning, females with vaginal plugs are considered to be at 0.5 days post-coitum.</li>
		<li>Euthanize the E17.5 pregnant females by cervical dislocation. Ensure that the animal is unresponsive by firmly pressing on the mouse paw (toe-pinch reflex). Wet the abdomen with 70% ethanol to sterilize and to prevent the spread of mouse fur.</li>
		<li>Make an incision in the skin at the middle of the abdomen using scissors and pull the skin apart with fingers.</li>
		<li>Open the abdominal cavity by pulling and making incisions in the abdominal muscle from the center towards the two sides of the abdomen using gross forceps and scissors. 
		NOTE: Be careful not to damage the small intestine, which is immediately below the peritoneal muscle.</li>
		<li>Collect the uterine horns (with the embryos) by cutting both at the level of the uterine body and of the ovaries. Soak them in a 90 x 15 mm2 Petri dish with 50 mL of DPBS.</li>
		<li>Isolate each embryo by cutting between each decidua, followed by the uterus muscular wall and the visceral yolk sac with a fine forceps and scissors. Pull out the embryos intact. Finally, cut the umbilical cord.</li>
		<li>Cut the E17.5 embryos&#39; heads with scissors (decapitation) before starting the dissection.</li>
	</ol>
	</li>
	<li>Collection of hearts
	<ol>
		<li>Soak the decapitated embryos in a 90 x 15 mm2 Petri dish containing a wet paper towel bedding in the dish bottom and 50 mL of HBSS with 1% FCS.</li>
		<li>Under a stereomicroscope, hold each embryo with gross forceps so that the ventral side is facing up.</li>
		<li>Carefully open the thoracic grid by cutting the right side of chest with scissors in order to prevent damage to the heart.</li>
		<li>Expose the heart by holding the rib cage with gross forceps.</li>
		<li>Pull apart the heart (still assembled with lungs and thymus) by holding the great vessels (containing the big vessels of in- and out-flow of the heart) and place them in a 35 x 15 mm2 Petri dish with 2 mL of HBSS 1% with FCS.</li>
		<li>Isolate the hearts from the surrounding organs and connective tissue with fine forceps and a scalpel.</li>
		<li>Wash the hearts in a new 35 x 15 mm Petri dish with 2 mL of HBSS 1% FCS and keep them on ice for 20 min to allow the heart to pump out the blood inside the chambers.</li>
	</ol>
	</li>
	<li>Preparation of heart cell suspensions
	<ol>
		<li>Pre-warm the 200 &#181;g/mL collagenase in HBSS+/+ (hereafter referred to as enzymatic solution) to 37 &#176;C. 
		NOTE: Dilute the collagenase in HBSS+/+ (with Ca2+ and Mg2+ cations) to maximize the enzyme activity; collagenase activity is stable between batches.</li>
		<li>Replace the washing solution soaking the hearts (HBSS 1% FCS) with 1 mL of pre-warmed enzymatic solution.</li>
		<li>Under a stereomicroscope, mince the hearts into 1-mm3 pieces using fine forceps and a scalpel. Add another 1 mL of pre-warmed enzymatic solution and transfer the minced tissue to a 5 mL tube with a lid. 
		NOTE: For an optimal digestion, place a maximum of 5 E17.5 hearts per 2 mL of enzymatic solution. If more hearts are digested at the same time, split them into different 5 mL tubes .</li>
		<li>Incubate the minced hearts for 15 min at 37 &#176;C. 
		NOTE: Lay down the 5 mL tubes (properly closed) in the incubator to spread the tissue along the solution.
		<ol>
			<li>At the end of the incubation, homogenize the tissue in the same enzymatic solution by pipetting up and down approximately 20 times with the P1000 pipette. Put the 5 mL tube in the vertical position and let the remaining tissue fragment sediment (approximately 3 min).</li>
			<li>Collect the supernatant (containing the digested cells in suspension) in a 50 mL tube, add 2 mL of HBSS 10% FCS (the same volume as the volume of enzymatic solution added to the tissue fragments), and keep them on ice while continuing the digestion process. 
			NOTE: The HBSS 10% FCS solution and the ice are important to inactive the enzyme action while the remaining tissue is digesting.</li>
			<li>Add 2 mL of pre-warmed enzymatic solution to the 5 mL tubes with the remaining tissue fragments and continue the digestion by repeating step 3.3.4 until no more tissue is observed. 
			NOTE: Around 4 cycles of digestion are enough to completely dissociate the heart tissue (5 E17.5 hearts in 2 mL of enzymatic solution).</li>
		</ol>
		</li>
		<li>Centrifuge the 50-mL tubes with cells in suspension for 10 min at 300 x g and discard the supernatant.</li>
		<li>Resuspend the cell pellet, add 1 mL of HBSS-/- 1% FCS, and filter the cell suspension though a 70 &#181;m nylon mesh. Count the cells with a Neubauer chamber using a Trypan blue vital stain. 
		NOTE: Resuspend the cells with HBSS-/- (without Ca2+ and Mg2+ cations) to minimize cell aggregation. Between 800,000 and 1,000,000 cells are obtained from one E17.5 heart.</li>
	</ol>
	</li>
	<li>Staining cardiac cells with fluorescent antibodies 
	NOTE: All antibodies are previously titrated to obtain optimal definition of the cell populations (the titration can vary with the antibody batch).The antibodies used in the heart panel are listed in Table 1.
	<ol>
		<li>Distribute the cardiac cells in suspension through a 96-well plate (round bottom). Distribute them evenly throughout all needed wells (200 &#181;L per well), short-spin centrifuge the 96-well plate for 1 min at 480 x g, and discard the supernatant. 
		NOTE: All wells of the 96-well plate (round bottom) can be used.</li>
		<li>Resuspend the pellet and incubate the cardiac cells for 20 min in the dark at 4 &#176;C with 100 &#181;L of the cocktail of antibodies (i.e., CD45-PE, TER119-PE, CD31-Alexa 647, and Sca-1-PE-Cyanine5) diluted in HBSS-/- 1% FCS.</li>
		<li>Wash the cardiac cells from the excess of antibodies by adding 200 &#181;L of HBSS-/- 1% FCS and short-spin centrifuging the cells for 1 min at 480 x g.</li>
		<li>Discard the supernatant and repeat the short-spin centrifugation (step 3.4.3).</li>
		<li>Resuspend the cells in 200 &#181;L of HBSS-/- 1% FCS and transfer them to a 5 mL tube already containing 200 &#181;L of HBSS-/- 1% FCS.</li>
		<li>Before acquisition, add 400 &#181;L of HBSS-/- 1% FCS with 0.5 &#181;g/mL PI and filter the cell suspension though a 70 &#181;m nylon mesh.</li>
	</ol>
	</li>
</ol>
4. Acquisition of Labeled Splenic, Intestinal, and Cardiac Cells with the Spectral Flow Cytometer
<ol>
	<li>Before acquisition of the data, automatically calibrate the spectral cytometer following the manufacturer&#39;s instructions. Perform the daily and the monthly quality control procedures each day of experiment.</li>
	<li>When the cytometer is ready, start the preview of the samples containing the cell suspensions labeled with all antibodies. 
	NOTE: Do not acquire.</li>
	<li>Make sure that all lasers are on as required. Adjust the FSC gain such that all cells are visible in the respective plots. Gate the cell population and apply this gate to the two spectral plots corresponding to the 488-nm and to the 638/405-nm lasers. 
	NOTE: When fluorochromes are excited by the 638-nm laser (i.e., when the 638-nm laser is on), there is a mask that shields light from 617-662 nm to prevent the 638 nm laser from shining into the PMT. This induces the loss of a part of the spectrum and therefore causes a lower separation of close dyes emitting in these wavelengths. However, the entire signal can be collected by switching off the 638-nm laser if it is not required for acquisition.</li>
	<li>Adjust the 32-channel photo multiplier (PMT) voltage (%) such that all fluorochromes are within the range of the intensity displayed in the y-axis (1-106) in the two different spectral plots displaying 488 nm and 405/638 nm excitations. 
	NOTE: Training is required to recognize the spectra of all fluorochromes used.</li>
	<li>Start to acquire the samples. Click on &#34;Preview&#34; to visualize the cells on the screen and then click on &#34;Acquire&#34; to record the sample. Save up to 2 x 106 events from each sample.</li>
	<li>After acquiring the stained samples, acquire the sample stained with the vital dye and then the compensation beads individually stained with all antibodies used. Click on &#8220;Preview&#8221; to visualize the cells on the screen and then on &#8220;Acquire&#8221; to record the samples. 
	NOTE: Keep the same PMT voltage for all samples and controls (i.e., beads and unstained samples); 5,000 events are largely sufficient.</li>
	<li>Finally, acquire the data from the unstained cell suspensions. Click on &#34;Preview&#34; to visualize the cells on the screen and then on &#34;Acquire&#34; to record the samples. 
	NOTE: Acquiring the unstained samples at the end of the experiment minimizes the carryover of fluorescent cells.</li>
	<li>Clean the cytometer following the manufacturer&#39;s instructions and close the experiment in the acquisition folder. 
	NOTE: Only after closing the experiment will it be &#160;saved and available for analysis.</li>
</ol>
5. Analysis of the Data with Spectral FCM Software
<ol>
	<li>In the &#34;Color Palette&#34; window of the &#34;Analysis&#34; tab, register all parameters present in the panel by adding each fluorochrome used and the corresponding marker (select from the available list or add a new parameter). Also, include the auto-fluorescence and viability parameters; all selected parameters will appear in the &#34;unmixing&#34; window.</li>
	<li>Select the first single stained sample (done with compensation beads) in the &#34;Control&#34; window under the &#34;Tube List&#34; of this experiment on the &#8220;Analysis&#8221; tab. In the worksheet, click on the &#34;polygon design tool&#34; and design a gate on the bead population in the FSC/SSC plot. Double-click on the top of gate to open a spectral or dot plot to visualize the gated beads in a daughter plot.
	<ol>
		<li>Design one gate for the positive and one gate for the negative beads either on the spectrum or on the dot plot. Check the whole spectra corresponding to the two laser sets for each positive and negative fraction and eliminate outliers by successively gating and clicking on the gate to verify the daughter population. Enter the negative and positive gates in the &#34;Unmixing&#34; window.</li>
		<li>Repeat step 5.2 for each single-stained sample. 
		NOTE: Be aware that the positive and negative gating strategy corresponds to the sample that is being analyzed, although the program allows for it to be placed in any sample. Instead of determining a negative fraction for each single-stained sample, use an unstained bead sample to set a universal negative.</li>
	</ol>
	</li>
	<li>Select the unstained cell sample in the &#34;Tube List&#34; and design gates for auto-fluorescent and for non-autofluorescent cells.</li>
	<li>When all the negative and positive gates for all parameters, including auto-fluorescence and viability, are selected in the &#34;Unmixing&#34; window, click on &#34;Calculate.&#34; Apply the unmixing to the samples to be analyzed.</li>
	<li>Analyze the data by designing gates and creating two-parameter plots.
	<ol>
		<li>First, design a gate for SSC versus FSC and then remove dead cells by excluding all cells labeled with the viability marker (PI versus CD45). On the rest of the cells, design gates for all populations of interest following the strategy described for each cell suspension (Figures 1-3). 
		NOTE: If necessary, adjust the compensation in the &#34;reference spectrum adjuster.&#34; This is a tool that can be used after unmixing to re-adjust the median of the positive populations for each fluorochrome if the algorithm could not do it perfectly.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Futamura, 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=Novel+full-spectral+flow+cytometry+with+multiple+spectrally-adjacent+fluorescent+proteins+and+fluorochromes+and+visualization+of+in+vivo+cellular+movement.">Novel full-spectral flow cytometry with multiple spectrally-adjacent fluorescent proteins and fluorochromes and visualization of in vivo cellular movement.</a> Cytometry A. 87 (9), 830-842 (2015).</li><li>Schmutz, S., Valente, M., Cumano, A., Novault, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectral+Cytometry+Has+Unique+Properties+Allowing+Multicolor+Analysis+of+Cell+Suspensions+Isolated+from+Solid+Tissues.">Spectral Cytometry Has Unique Properties Allowing Multicolor Analysis of Cell Suspensions Isolated from Solid Tissues.</a> PLoS One. 11 (8), e0159961 (2016).</li><li>Roederer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiparameter+FACS+analysis.">Multiparameter FACS analysis.</a> Curr Protoc Immunol. , (2002).</li><li>Perfetto, S. P., Chattopadhyay, P. K., Roederer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seventeen-colour+flow+cytometry:+unravelling+the+immune+system.">Seventeen-colour flow cytometry: unravelling the immune system.</a> Nat Rev Immunol. 4 (8), 648-655 (2004).</li><li>Grigoriadou, K., Boucontet, L., Pereira, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+receptor-gamma+allele-specific+selection+of+V+gamma+1/V+delta+4+cells+in+the+intestinal+epithelium.">T cell receptor-gamma allele-specific selection of V gamma 1/V delta 4 cells in the intestinal epithelium.</a> J Immunol. 169 (7), 3736-3743 (2002).</li><li>Guy-Grand, D., 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=Origin,+trafficking,+and+intraepithelial+fate+of+gut-tropic+T+cells.">Origin, trafficking, and intraepithelial fate of gut-tropic T cells.</a> J Exp Med. 210 (9), 1839-1854 (2013).</li><li>Lavoinne, A., Cauliez, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=[Cardiac+troponin+I+and+T:+specific+biomarkers+of+cardiomyocyte].">[Cardiac troponin I and T: specific biomarkers of cardiomyocyte].</a> Rev Med Interne. 25 (2), 115-123 (2004).</li><li>Staudt, D. W., Liu, J., Thorn, K. S., Stuurman, N., Liebling, M., Stainier, D. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+imaging+of+cardiomyocyte+behavior+reveals+two+distinct+steps+in+ventricular+trabeculation.">High-resolution imaging of cardiomyocyte behavior reveals two distinct steps in ventricular trabeculation.</a> Development. 141 (3), 585-593 (2014).</li><li>Beltrami, A. 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=Adult+cardiac+stem+cells+are+multipotent+and+support+myocardial+regeneration.">Adult cardiac stem cells are multipotent and support myocardial regeneration.</a> Cell. 114 (6), 763-776 (2003).</li><li>Keith, M. C., Bolli, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term="String+theory"+of+c-kit(pos)+cardiac+cells:+a+new+paradigm+regarding+the+nature+of+these+cells+that+may+reconcile+apparently+discrepant+results.">"String theory" of c-kit(pos) cardiac cells: a new paradigm regarding the nature of these cells that may reconcile apparently discrepant results.</a> Circ Res. 116 (7), 1216-1230 (2015).</li><li>Valente, M., Nascimento, D. S., Cumano, A., Pinto-do-&#211;, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sca-1++cardiac+progenitor+cells+and+heart-making:+a+critical+synopsis.">Sca-1+ cardiac progenitor cells and heart-making: a critical synopsis.</a> Stem Cells Dev. 23 (19), 2263-2273 (2014).</li></ol>

The authors have nothing to disclose.

Conventional FCM is based on the detection of photons emitted after the excitation of fluorescent probes. The fluorescence emission of one fluorochrome detected in a detector designed to measure the signal from another fluorochrome induces physical overlap. This spillover among emission spectra needs to be corrected by compensations.
Spectral FCM and data processing by the unmixing deconvolution algorithm allows for the combination of fluorochromes with close emission peaks without additional compensation, provided that they have different spectral shapes. The algorithm used for the analysis treats auto-fluorescence as an independent parameter, subtracting non-specific fluorescence from solid tissue cell suspensions.
A panel of 19 fluorochromes was assembled to analyze immune cells using only two lasers (488 nm and 405 nm). Indeed, when the 638 nm laser was on, a section of the 32-channel PMT corresponding to the excitation wavelength of this laser was not available. To get the maximum detection capacity of the PMT, the red laser was switched off. Mouse splenic cells were analyzed, allowing for the detection of more than 12 different cell populations, some of which comprised 1% or less of the hematopoietic cells.
To test the capacity of spectral FCM to manage auto-fluorescence, two different situations that can impair the analysis were tested: one where lymphocytes were contaminated with epithelial cells, and one where auto-fluorescent cardiac cells were analyzed. In contrast with conventional FCM, spectral cytometry allows for the discrimination of all lymphocyte subsets, even when they represent minor subsets.
The controversy around the regenerative capacity of adult cardiomyocytes9,10,11 is in part due to the absence of a strategy to discriminate and isolate distinct subsets of viable cells in the heart. A multi-parametric analysis defining unique combinations of markers to discriminate the different cardiac subsets might enable the detection of rare subsets because of large numbers of cells that can be analyzed. Fetal hearts (E17.5) were analyzed, and a large fraction of auto-fluorescent cells, which would have escaped detection in conventional FCM, was integrated in the non-hematopoietic viable cardiac cell compartment in spectral FCM.
A recurrent problem of this method is the requirement for good-quality fluorescent labeled antibodies. This is particularly critical when using tandem dyes, where a component of the tandem is dominant. For example, BV786 and Cy7-PE can be difficult to distinguish from BV421 and PE, respectively.
FCM based on spectrometry provides analytical power higher than that obtained with conventional FCM, without the need for compensation matrices. This manuscript provides evidence that there is an alternative approach that demonstrates a high analytical capacity, the absence of complex compensation matrices, the unimpaired discrimination of cell subsets due to auto-fluorescence, and no additional financial commitments that still required for mass spectrometry FCM. Spectral FCM thus appears to be a method of choice for the analysis of cells obtained from a wide variety of solid tissues usually not amenable to conventional FCM and can be used in tumor, developmental, and stem cell biology.

In the last few decades, flow cytometry (FCM) became a widely available analytical method essential for cell phenotyping studies. There has been a substantial increase in the available fluorescent dyes, particularly fluorochromes excited by the violet laser (405 nm) (e.g., Brilliant Violet and new Q-dot dyes). However, the growth of available fluorescent dyes increases the risk of overlapping emissions and requires labor-intensive compensation matrices. FCM became widely used to analyze cell suspensions from solid tissue, but the presence of auto-fluorescent cells limits the discrimination of specifically labeled populations.
The basic principles of spectral FCM are reported in detail in Futamura et al.1,2. Briefly, the spectral FCM used here (see the table of materials) is equipped with 405, 488, and 638 nm lasers. The spectral FCM captures all the emitted fluorescence as spectra in a 32-channel linear array PMT (32ch PMT) for 500 nm to 800 nm and 2 independent PMTs for 420 nm to 440 nm and 450 nm to 469 nm, respectively, which replace the conventional band-pass filters. The 488 and the 405/638 nm laser spots are spatially separated, while the 405 nm and 638 nm laser spots are co-linear. For each individual particle, the spectral FCM measures up to 66 channels of fluorescence data excited by the 405 nm and 488 nm lasers. When cells are excited by the 638 nm laser, the spectral FCM measures 58 channels of fluorescence data because a mask is inserted to prevent the 638 nm laser from shining into the PMT. Spectral FCM analyzes the acquired full spectrum data with an algorithm based on the weighted least squares method (WLSM), which enables the separation of overlapping fluorescent spectra. Spectra derived from single-stained and unstained samples are recognized as the basic reference spectra. Multi-stained samples are mathematically fitted and unmixed, and the spectrum of a sample with mixed fluorescent labels is decomposed into a collection of its constituent spectra. The unmixing, in spectral technology3,4, replaces the compensation that removes the signal from all detectors except the one measuring a given dye.
In this study, we combined and tested 19 fluorochromes in a single, 21-parameter analysis that characterized the major hematopoietic subsets found in the mouse spleen. Additionally, we demonstrated that the spectral cytometry can manage auto-fluorescent signal, thus improving the characterization of intestinal intra-epithelial lymphocytes and of the embryonic heart. Indeed, in these tissues, auto-fluorescence management allowed for the assignment of specific fluorescence to cells that would be excluded from the analysis in conventional FCM.

<table><tbody><tr><td>Mice</td><td>Janvier Labs</td><td>CD45.2</td><td>Source of cells</td></tr><tr><td>Ethanol 70%</td><td>VWR</td><td>83801.36</td><td>Sterilization</td></tr><tr><td>DPBS (Ca&lt;sup&gt;2+&lt;/sup&gt;, Mg&lt;sup&gt;2+&lt;/sup&gt;)</td><td>GIBCO ThermoFisher</td><td>14040-174</td><td>Embryos collection</td></tr><tr><td>Hanks&#039; Balanced Salt Solution (+Ca&lt;sup&gt;2+&lt;/sup&gt; +Mg&lt;sup&gt;2+&lt;/sup&gt;) (HBSS+/+)</td><td>GIBCO Life ThermoFisher</td><td>14025092</td><td>Dissociation, digestion and staining solutions</td></tr><tr><td>Hanks&#039; Balanced Salt Solution (-Ca&lt;sup&gt;2+&lt;/sup&gt;, -Mg&lt;sup&gt;2+&lt;/sup&gt;) (HBSS-/-)</td><td>GIBCO Life ThermoFisher</td><td>14175095</td><td>Dissociation, digestion and staining solutions</td></tr><tr><td>Fetal calf serum (FCS)</td><td>EUROBIO</td><td>CVFSVF00-0U</td><td>Dissociation, digestion and staining solutions</td></tr><tr><td>Collagenase</td><td>Sigma</td><td>C2139</td><td>Enzymatic solution</td></tr><tr><td>90 x 15 mm,&lt;br/&gt; plastic tissue culture Petri dishes.</td><td>TPP</td><td>T93100</td><td>Embryos and hearts collection</td></tr><tr><td>35 x 15 mm,&lt;br/&gt; plastic tissue culture Petri dishes.</td><td>TPP</td><td>T9340</td><td>Hearts collection</td></tr><tr><td>Fine iris scissor</td><td>Fine Science Tools</td><td>14090-09</td><td>Dissection tools</td></tr><tr><td>Gross forceps (narrow pattern forceps, curved 12 cm)</td><td>Fine Science Tools</td><td>11003-12</td><td>Dissection tools</td></tr><tr><td>Fine forceps (Dumont no. 7 forceps)</td><td>Fine Science Tools</td><td>11272-30</td><td>Dissection tools</td></tr><tr><td>Scalpel&amp;nbsp;</td><td>VWR</td><td>21909-668</td><td>Dissection tools</td></tr><tr><td>LEICA MZ6 Dissection microscope</td><td>LEICA</td><td>MZ6 10445111</td><td>Sampling; Occular W-Pl10x/23</td></tr><tr><td>Cold lamp source</td><td>SCHOTT VWR</td><td>KL1500 compact</td><td>Sampling;&lt;br/&gt; Two goose neck fibers adapted</td></tr><tr><td>FACS tubes</td><td>VWR</td><td>60819-310</td><td>Digesting cells</td></tr><tr><td>96 well plate (round bottom)</td><td>VWR</td><td>10861-564</td><td>Staining cells</td></tr><tr><td>Nylon mesh bolting cloth sterilized,&lt;br/&gt; 50/50 mm pieces.</td><td>SEFAR NITEX</td><td>SEFAR NITEX</td><td>Cell filtration</td></tr><tr><td>UltrasComp eBeads</td><td></td><td></td><td></td></tr><tr><td>Fluorescence labeled antibodies</td><td>Biolegend</td><td>Catalogue number see table below</td><td>Staining cells</td></tr></tbody></table>

analysis of cell suspensions isolated from solid tissues by spectral flow cytometry

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to the analysis of a few fluorochromes, currently there are dozens of different fluorescent dyes, and up to 14-18 different dyes can be combined at a time. However, several limitations still impair the analytical capabilities. Because of the multiplicity of fluorescent probes, data analysis has become increasingly complex due to the need of large, multi-parametric compensation matrices. Moreover, mutant mouse models carrying fluorescent proteins to detect and trace specific cell types in different tissues have become available, so the analysis (by flow cytometry) of auto-fluorescent cell suspensions obtained from solid organs is required. Spectral flow cytometry, which distinguishes the shapes of emission spectra along a wide range of continuous wavelengths, addresses some of these problems. The data is analyzed with an algorithm that replaces compensation matrices and treats auto-fluorescence as an independent parameter. Thus, spectral flow cytometry should be capable of discriminating fluorochromes with similar emission peaks and can provide a multi-parametric analysis without compensation requirements.
This protocol describes the spectral flow cytometry analysis, allowing for a 21-parameter (19 fluorescent probes) characterization and the management of an auto-fluorescent signal, providing high resolution in minor population detection. The results presented here show that spectral flow cytometry presents advantages in the analysis of cell populations from tissues difficult to characterize in conventional flow cytometry, such as the heart and the intestine. Spectral flow cytometry thus demonstrates the multi-parametric analytical capacity of high-performing conventional flow cytometry without the requirement for compensation and enables auto-fluorescence management.

21-parameter spectral FCM panel to analyze splenocytes
Figure 1 shows the results obtained with the 19-fluorescent-antibody panel applied to splenic cells comprising different antibodies recognizing subsets of T, B, NK, dendritic, and myeloid cells, while CD45 labels all hematopoietic nucleated cells. The panel also includes a viability dye (PI), as well as the size (FSC) and granularity (SSC) parameters. Figure 1A shows the reference fluorochrome spectra. After the elimination of dead cells in the CD45+ gate (Figure 1B), the analysis shows that CD3+ T cells express the TcR&#946; chain and have the expected CD4/CD8 distribution and proportion of CD25+ cells in CD4 T cells. CD8 T cells show a normal distribution of CD44- and Ly49D-expressing cells. CD19+ B cells co-express B220 and MHCII, as well as IgM and IgD, typical of mature splenic B cells. A subset of NK1.1+ NK cells represents a small subset of the splenic lymphocytes that co-express Ly49D. The remaining cells comprise a small subset of CD11c+ dendritic cells with high levels of MHC II, some of which were also CD11b+. F4/80+ cells characteristic of tissue-resident macrophages co-express CD11b and the highest levels of CD44 found in the spleen. The remaining cells (CD19-Nk1.1-CD3-F4/80-CD11c-) could be subdivided by the expression of CD11b and Gr1, where the small subset of double-positive cells also express high levels of CD44, likely corresponding to progenitors known to be present at low frequencies in the spleen. A large fraction was negative for the three markers. Figure 2 shows the same analysis before adjusting the compensation.
<img alt="Figure 1" src="/files/ftp_upload/55578/55578fig1.jpg" /> 
Figure 1: A 19-color Antibody Panel for the Analysis of Murine Spleen Cells. (A) Mouse splenocytes were stained with the previous antibody combination, to which CD45-evolve 655 was added. The reference spectra of all fluorochromes are shown. Fluorophore-labeled CD45 is marked by a red arrow. The fluorochromes are listed in Table 1. (B) The contour plots show the discriminative capacity of this multi-parametric analysis to separate the different populations of B, T, NK, dendritic, or myeloid cells. The analysis was done in conventional FCM software after unmixing deconvolution.
<img alt="Figure 2" src="/files/ftp_upload/55578/55578fig2.jpg" /> 
Figure 2: A 19-color Antibody Panel for the Analysis of Murine Spleen Cells. The same analysis as in Figure 1 was done before the adjustment of the compensation with the reference spectra adjuster.
Small intestine intra-epithelial lymphocytes
Several T-cell populations are found in the intestine within the epithelial cell layer5,6. Conventional isolation of these subsets includes a density gradient that separates lymphocytes from epithelial cells. However, this preparation is delicate, time consuming, and results in cell loss. To improve quantification and to facilitate the experimental procedure, the potential of spectral FCM to discriminate populations of intestinal lymphocytes within a large fraction of epithelial cells was tested. After the mechanical disruption of the epithelial tissue, cell suspensions were stained with antibodies that label subsets of &#945;&#946; and &#947;&#948; T cells usually found in the intestinal epithelium. The cells were analyzed in two conventional cytometers and in spectral FCM.
Figure 3 shows the results after following a similar gating strategy, with elimination of dead cells gating on PI-negative cells. The presence of auto-fluorescent cells is obvious in all conventional cytometers and in spectral cytometry when the auto-fluorescence manager is inactivated (top panels, red arrows), resulting in a poor discrimination of the living cells. In all plots, cells within the FSC-A/SSC-A lymphocyte gate are shown in blue and cells with larger scatters (epithelial cell gate) are shown in red. Both conventional instruments were unable to resolve the CD3+ T-cell subset (in blue) (turquoise arrow) from the remaining cells. Therefore, TcR&#946;- cells (green arrow) contaminated the CD3+TcR&#947;&#948;- population, a biologically improbable subset never detected after the separation of the lymphocytes from epithelial cells. In contrast, in spectral FCM after auto-fluorescence management, the analysis of the different T-cell subsets was not impaired by the presence of epithelial cells, the populations were well-separated, and there were no TcR-negative cells in the CD3+ gate.
<img alt="Figure 3" src="/files/ftp_upload/55578/55578fig3.jpg" /> 
Figure 3: The Presence of Auto-fluorescent Cells Does not Impair the Detection of Intra-epithelial Lymphocytes in Spectral FCM. Small intestinal cells, including epithelial cells and lymphocytes, were stained with antibodies recognizing TcR&#948;, PI, TcR&#946; Cy7-APC CD3 (panels B), V&#947;7 APC, V&#948;4 Cy7-PE (panels C), and CD8 FITC (panels D). PI was added in the FACS buffer before analysis. The acquisition of the data was done sequentially in the three instruments after the appropriate quality control. Doublets were eliminated in FSC-H/FSC-W. The analysis was done in conventional FCM software. Plots show the different steps of the gating strategy. Cells within the FSC-A/SSC-A lymphocyte gate are labeled in blue, while cells within the intestinal epithelial cell gate are labeled in red. Arrows point to different population distortions and auto-fluorescence. Plots in C show the analysis of data obtained in spectral FCM without autofluorescence management in the unmixing, whereas D shows the same data after unmixing with autofluorescence management.
Embryonic heart
Conventional FCM showed a major population of auto-fluorescent cells (black arrow) that was excluded from the analysis because it fluoresced in the CD45 and TER119 dump channel (PE), marking hematopoietic cells. In contrast, this auto-fluorescent population was not present as such in spectral FCM and was comprised in the CD45-TER119- subset (Figure 4). To show that these cells were cardiac and not endothelial or hematopoietic, expressing low levels of CD45, TER119, or CD31, the expression of transcripts specific to cardiomyocytes were quantified on sorted cells. High levels of cardiac muscle troponin T (TNNT2)7 and atrial light chain-2 (MYL7)8 transcripts were found in the auto-fluorescent population.
<img alt="Figure 4" src="/files/ftp_upload/55578/55578fig4.jpg" /> 
Figure 4: Auto-fluorescent Cells in E17.5 Cardiac Cell Suspensions are Properly Included in the Negative Cell Fraction in Spectral FCM. (A) Cell suspensions from fetal hearts were stained with anti-TER119 and CD45-PE, Sca-1-PECy5, and CD31-APC antibodies and sequentially acquired in the three instruments. Black arrows show auto-fluorescent cells in the two conventional cytometers, while these cells are not detected as auto-fluorescent in spectral cytometry and can thus be analyzed for specific fluorescent staining. (B) Auto-fluorescent cells (within the elliptical gate in green), but not CD31+ cells (rectangular gate in blue, negative control), showed the expression of cardiac troponin (TNNT2)7 and atrial light chain-2 (MYL7)8 transcripts. a.u. &#8211; arbitrary units relative to the GAPDH house-keeping transcript. Data presented as mean value &#177; SEM.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Excitation laser</td>
			<td>Fluorochrome</td>
			<td>Specificity</td>
			<td>Clone</td>
			<td></td>
		</tr>
		<tr>
			<td>488 nm</td>
			<td>BB515</td>
			<td>CD8</td>
			<td>53-6.7</td>
			<td></td>
		</tr>
		<tr>
			<td>488 nm</td>
			<td>FITC</td>
			<td>Ly49D</td>
			<td>4E5</td>
			<td></td>
		</tr>
		<tr>
			<td>488 nm</td>
			<td>PE</td>
			<td>F4/80</td>
			<td>6F12</td>
			<td></td>
		</tr>
		<tr>
			<td>488 nm</td>
			<td>PE-CF594</td>
			<td>NK1.1</td>
			<td>PK136</td>
			<td></td>
		</tr>
		<tr>
			<td>488 nm</td>
			<td>PE-Cy5</td>
			<td>CD44</td>
			<td>IM7</td>
			<td></td>
		</tr>
		<tr>
			<td>488 nm</td>
			<td>PE-Cy7</td>
			<td>CD11b</td>
			<td>M1/70</td>
			<td></td>
		</tr>
		<tr>
			<td>488 nm</td>
			<td>PerCP-Cy5.5</td>
			<td>IgM</td>
			<td>R6-60.2</td>
			<td></td>
		</tr>
		<tr>
			<td>488 nm</td>
			<td>PerCP</td>
			<td>Gr-1</td>
			<td>RB6-8CS</td>
			<td></td>
		</tr>
		<tr>
			<td>488 nm</td>
			<td>AF532</td>
			<td>CD3</td>
			<td>17A2</td>
			<td></td>
		</tr>
		<tr>
			<td>488 nm</td>
			<td>Propidium Iodide</td>
			<td>Viability</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>405 nm</td>
			<td>V450</td>
			<td>IgD</td>
			<td>11-26c.2a</td>
			<td></td>
		</tr>
		<tr>
			<td>405 nm</td>
			<td>BV421</td>
			<td>CD11c</td>
			<td>HL3</td>
			<td></td>
		</tr>
		<tr>
			<td>405 nm</td>
			<td>BV510</td>
			<td>CD19</td>
			<td>1D3</td>
			<td></td>
		</tr>
		<tr>
			<td>405 nm</td>
			<td>BV570</td>
			<td>TCRb</td>
			<td>H57-597</td>
			<td></td>
		</tr>
		<tr>
			<td>405 nm</td>
			<td>BV605</td>
			<td>CD4</td>
			<td>RM4-5</td>
			<td></td>
		</tr>
		<tr>
			<td>405 nm</td>
			<td>BV650</td>
			<td>CD45R/B220</td>
			<td>RA3-6B2</td>
			<td></td>
		</tr>
		<tr>
			<td>405 nm</td>
			<td>BV711</td>
			<td>I-A/I-E</td>
			<td>M5/114</td>
			<td></td>
		</tr>
		<tr>
			<td>405 nm</td>
			<td>BV786</td>
			<td>CD25</td>
			<td>PC61</td>
			<td></td>
		</tr>
		<tr>
			<td>405 nm</td>
			<td>eVolve 655</td>
			<td>CD45</td>
			<td>30-F11</td>
			<td></td>
		</tr>
		<tr>
			<td>N/A</td>
			<td>purified</td>
			<td>CD16/CD32</td>
			<td>2.4G2</td>
			<td>Fc-receptor block</td>
		</tr>
		<tr>
			<td colspan="3">List of Antibodies used in cardiac cell suspension</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>638 nm</td>
			<td>Alexa Fluor 647</td>
			<td>CD31</td>
			<td>MEC13.3</td>
			<td></td>
		</tr>
		<tr>
			<td>488 nm</td>
			<td>PE</td>
			<td>CD45</td>
			<td>30-F11</td>
			<td></td>
		</tr>
		<tr>
			<td>488 nm</td>
			<td>PE-Cy5</td>
			<td>Sca-1</td>
			<td>D7</td>
			<td></td>
		</tr>
		<tr>
			<td>488 nm</td>
			<td>PE</td>
			<td>Ter119</td>
			<td>TER-119</td>
			<td></td>
		</tr>
	</tbody>
</table>
Table1: List of Antibodies Used in the 19-color Panel and in Cardiac Cell Suspension.

Watch this Scientific Journal Video about Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry at JoVE.com

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

This article describes spectral cytometry, a new approach in flow cytometry that uses the shapes of emission spectra to distinguish fluorochromes. An algorithm replaces compensations and can treat auto-fluorescence as an independent parameter. This new approach allows for the proper analysis of cells isolated from solid organs.

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to ...

analysis-cell-suspensions-isolated-from-solid-tissues-spectral-flow

Nanyang Technological University

Research

JoVE Journal

Biology

12.9K Views.  Institut Pasteur.  Dit artikel beschrijft spectrale cytometry, een nieuwe benadering flowcytometrie dat de vormen van emissiespectra gebruikt om fluorochromen onderscheiden. Een algoritme vervangt vergoedingen en kan de behandeling van auto-fluorescentie als een onafhankelijke parameter. Deze nieuwe aanpak maakt het mogelijk voor een juiste analyse van de cellen geïsoleerd van vaste organen. 

Video: Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

Processing of Primary Brain Tumor Tissue for Stem Cell Assays and Flow Sorting

Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction of cells in the tumor with stem cell properties, termed brain tumor initiating cells (BTICs), possess an ability to differentiate along multiple lineages, self-renew, and initiate tumors in vivo. We applied culture conditions originally used for normal neural stem cells (NSCs) to a variety of human brain tumors and found that this culture method specifically selects for stem-like populations. Serum-free medium (NSC) allows for the maintenance of an undifferentiated stem cell state, and the addition of bFGF and EGF allows for the proliferation of multi-potent, self-renewing, and expandable tumorspheres.
To further characterize each tumor's BTIC population, we evaluate cell surface markers by flow cytometry. We may also sort populations of interest for more specific characterization. Self-renewal assays are performed on single BTICs sorted into 96 well plates; the formation of tumorspheres following incubation at 37 &deg;C indicates the presence of a stem or progenitor cell. Multiple cell numbers of a particular population can also be sorted in different wells for limiting dilution analysis, to analyze self-renewal capacity. We can also study differential gene expression within a particular cell population by using single cell RT-PCR.
The following protocols describe our procedures for the dissociation and culturing of primary human samples to enrich for BTIC populations, as well as the dissociation of tumorspheres. Also included are protocols for staining for flow cytometry analysis or sorting, self-renewal assays, and single cell RT-PCR.

The identification of brain tumor initiating cells (BTICs), the rare cells within a heterogeneous tumor possessing stem cell properties, provides new insights into human brain tumor pathogenesis. We have refined specific culture conditions to enrich for BTICs, and we routinely use flow cytometry to further enrich these populations. Self-renewal assays and transcript analysis by single cell RT-PCR can subsequently be performed on these isolated cells.

Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction ...

Medicine

Characterizing Microbiome Dynamics &#8211; Flow Cytometry Based Workflows from Pure Cultures to Natural Communities

The investigation of pure cultures and monitoring of microbial community dynamics is vital to understand and control natural ecosystems and technical applications driven by microorganisms. Next generation sequencing methods are widely utilized to resolve microbiomes, but they are generally resource and time intensive and deliver mostly qualitative information. Flow cytometric microbiome analysis does not suffer from those disadvantages and can provide relative subcommunity abundances and absolute cell numbers at-line. Although it does not deliver direct phylogenetic information, it can enhance the analysis depth and resolution of sequencing approaches. In sharp contrast to medical applications in both research and routine settings, flow cytometry is still not widely used for microbiome analysis. Missing information on sample preparation and data analysis pipelines may create an entry barrier for the researchers facing microbiome analysis challenges that would often be textbook flow cytometry applications. Here, we present three comprehensive workflows for pure cultures, complex communities in clear medium and complex communities in challenging matrices, respectively. We describe individual sampling and fixation procedures and optimized staining protocols for the respective sample sets. We elaborate the cytometric analysis with a complex research centered and an application focused bench top device, describe the cell sorting procedure and suggest data analysis packages. We furthermore propose important experimental controls and apply the presented workflows to the respective sample sets.

Flow cytometric analysis has proven valuable for investigating pure cultures and monitoring microbial community dynamics. We present three comprehensive workflows, from sampling to data analysis, for pure cultures and complex communities in clear medium as well as in challenging matrices, respectively.

The investigation of pure cultures and monitoring of microbial community dynamics is vital to understand and control natural ecosystems and technical ...

Environment

Flow Cytometry Protocols for Surface and Intracellular Antigen Analyses of Neural Cell Types

Flow cytometry has been extensively used to define cell populations in immunology, hematology and oncology. Here, we provide a detailed description of protocols for flow cytometric analysis of the cluster of differentiation (CD) surface antigens and intracellular antigens in neural cell types. Our step-by-step description of the methodological procedures include: the harvesting of neural in vitro cultures, an optional carboxyfluorescein succinimidyl ester (CFSE)-labeling step, followed by surface antigen staining with conjugated CD antibodies (e.g., CD24, CD54), and subsequent intracellar antigen detection via primary/secondary antibodies or fluorescently labeled Fab fragments (Zenon labeling). The video demonstrates the most critical steps. Moreover, principles of experimental planning, the inclusion of critical controls, and fundamentals of flow cytometric analysis (identification of target population and exclusion of debris; gating strategy; compensation for spectral overlap) are briefly explained in order to enable neurobiologists with limited prior knowledge or specific training in flow cytometry to assess its utility and to better exploit this powerful methodology.

We provide a detailed description of a protocol for flow cytometric analysis of surface antigens and/or intracellular antigens in neural cell types. Critical aspects of experimental planning, step-by-step methodological procedures, and fundamental principles of flow cytometry are explained in order to enable neurobiologists to exploit this powerful technology.

Flow cytometry has been extensively used to define cell populations in immunology, hematology and oncology. Here, we provide a detailed description of ...

Neuroscience

Analysis of Immune Cells in Single Sciatic Nerves and Dorsal Root Ganglion from a Single Mouse Using Flow Cytometry

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of the PNS are affected by injury and disease, affecting the nerve function and the capacity for regeneration. Neuronal immune cells are commonly analyzed by immunofluorescence (IF). While IF is essential for determining the location of the immune cells in the nerve, IF is only semi-quantitative and the method is limited to the number of markers that can be analyzed simultaneously and the degree of surface expression. In this study, flow cytometry was used for quantitative analysis of leukocyte infiltration into sciatic nerves or dorsal root ganglions (DRGs) of individual mice. Single cell analysis was performed using DAPI and several proteins were analyzed simultaneously for either surface or intracellular expression. Both sciatic nerves from one mouse that were treated according to this protocol generated &#8805; 30,000 single nucleated events. The proportion of leukocytes in the sciatic nerves, determined by expression of CD45, was approximately 5% of total cell content in the sciatic nerve and approximately 5-10% in the DRG. Although this protocol focuses primarily on the immune cell population within the PNS, the flexibility of flow cytometry to measure a number of markers simultaneously means that the other cells populations present within the nerve, such as Schwann cells, pericytes, fibroblasts, and endothelial cells, can also be analyzed using this method. This method therefore provides a new means for studying systemic effects on the PNS, such as neurotoxicology and genetic models of neuropathy or in chronic diseases, such as diabetes.

Quantitative analysis of cell content within the murine sciatic nerve is difficult due to the scarcity of the tissue. This protocol describes a method for tissue digestion and preparation that provides sufficient cells for flow cytometry analysis of immune cell populations from nerves of individual mice.

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of ...

Determination of Regulatory T Cell Subsets in Murine Thymus, Pancreatic Draining Lymph Node and Spleen Using Flow Cytometry

Our immune system consists of a number and variety of immune cells including regulatory T cells (Treg) cells. Treg cells can be divided into two subsets, thymic derived Treg (tTreg) cells and peripherally induced Treg (pTreg) cells. They are present in different organs of our body and can be distinguished by specific markers, such as Helios and Neuropilin 1. It has been reported that tTreg cells are functionally more suppressive than pTreg cells. Therefore, it is important to determine the proportion of both tTreg and pTreg cells when investigating heterogeneous cell populations. Herein, we collected thymic glands, pancreatic draining lymph nodes and spleens from normoglycemic non-obese diabetic mice to distinguish tTreg cells from pTreg cells using flow cytometry. We manually prepared single cell suspensions from these organs. Fluorochrome conjugated surface CD4, CD8, CD25, and Neuropilin 1 antibodies were used to stain the cells. They were kept in the fridge overnight. On the next day, the cells were stained with fluorochrome conjugated intracellular Foxp3 and Helios antibodies. These markers were used to characterize the two subsets of Treg cells. This protocol demonstrates a simple but practical way to prepare single cells from murine thymus, pancreatic draining lymph node and spleen and use them for subsequent flow cytometric analysis.

Herein, we present a protocol to prepare single cells from murine thymus, pancreatic draining lymph node and spleen to further study these cells using flow cytometry. In addition, this protocol was used for determining the subsets of regulatory T cells using flow cytometry.

Our immune system consists of a number and variety of immune cells including regulatory T cells (Treg) cells. Treg cells can be divided into two subsets, ...

Immunology and Infection

High-Dimensionality Flow Cytometry for Immune Function Analysis of Dissected Implant Tissues

The success of implanting laboratory-grown tissue or a medical device in an individual is subject to the immune response of the recipient host. Considering an implant as a foreign body, a hostile and dysregulated immune response may result in the rejection of the implant, while a regulated response and regaining of homeostasis can lead to its acceptance. Analyzing the microenvironments of implants dissected out under in vivo or ex vivo&#160;settings can help in understanding the pattern of immune response, which can ultimately help in developing new generations of biomaterials. Flow cytometry is a well-known technique for characterizing immune cells and their subsets based on their cell surface markers. This review describes a protocol based on manual dicing, enzymatic digestion, and filtration through a cell strainer for the isolation of uniform cell suspensions from dissected implant tissue. Further, a multicolor flow cytometry staining protocol has been explained, along with steps for initial cytometer settings to characterize and quantify these isolated cells by flow cytometry.

Isolation of cells from dissected implants and their characterization by flow cytometry can significantly contribute to understanding the pattern of immune response against implants. This paper describes a precise method for the isolation of cells from dissected implants and their staining for flow cytometric analysis.

The success of implanting laboratory-grown tissue or a medical device in an individual is subject to the immune response of the recipient host. ...

Bioengineering

Simple and Efficient Technique for the Preparation of Testicular Cell Suspensions

Mammalian testes are very complex organs that contain over 30 different cell types, including somatic testicular cells and different stages of germline cells. This heterogeneity is an important drawback concerning the study of the bases of mammalian spermatogenesis, as pure or enriched cell populations in certain stages of sperm development are needed for most molecular analyses1.
 Various strategies such as Staput2,3, centrifugal elutriation1, and flow cytometry (FC)4,5 have been employed to obtain enriched or purified testicular cell populations in order to enable differential gene expression studies. 
It is required that cells are in suspension for most enrichment/ purification approaches. Ideally, the cell suspension will be representative of the original tissue, have a high proportion of viable cells and few multinucleates - which tend to form because of the syncytial nature of the seminiferous epithelium6,7 - and lack cell clumps1 . Previous reports had evidenced that testicular cell suspensions prepared by an exclusively mechanical method clumped more easily than trypsinized ones1 . On the other hand, enzymatic treatments with RNAses and/or disaggregating enzymes like trypsin and collagenase lead to specific macromolecules degradation, which is undesirable for certain downstream applications. The ideal process should be as short as possible and involve minimal manipulation, so as to achieve a good preservation of macromolecules of interest such as mRNAs. Current protocols for the preparation of cell suspensions from solid tissues are usually time-consuming, highly operator-dependent, and may selectively damage certain cell types1,8 . 
	The protocol presented here combines the advantages of a highly reproducible and extremely brief mechanical disaggregation with the absence of enzymatic treatment, leading to good quality cell suspensions that can be used for flow cytometric analysis and sorting4, and ulterior gene expression studies9 .

A novel protocol for the mechanical preparation of testicular cell suspensions from rodent material, avoiding enzymes and detergents, is described. The method is very simple, fast, reproducible, and renders good quality cell suspensions, which are suitable for flow sorting and RNA extraction.

Mammalian testes are very complex organs that contain over 30 different cell types, including somatic testicular cells and different stages of germline ...

Simultaneous Flow Cytometric Characterization of Multiple Cell Types Retrieved from Mouse Brain/Spinal Cord Through Different Homogenization Methods

Recent advances in viral vector and nanomaterial sciences have opened the way for new cutting-edge approaches to investigate or manipulate the central nervous system (CNS). However, further optimization of these technologies would benefit from methods allowing rapid and streamline determination of the extent of CNS and cell-specific targeting upon administration of viral vectors or nanoparticles in the body. Here, we present a protocol that takes advantage of the high throughput and multiplexing capabilities of flow cytometry to allow a straightforward quantification of different cell subtypes isolated from mouse brain or spinal cord, namely microglia/macrophages, lymphocytes, astrocytes, oligodendrocytes, neurons and endothelial cells. We apply this approach to highlight critical differences between two tissue homogenization methods in terms of cell yield, viability and composition. This could instruct the user to choose the best method depending on the cell type(s) of interest and the specific application. This method is not suited for analysis of anatomical distribution, since the tissue is homogenized to generate a single-cell suspension. However, it allows to work with viable cells and it can be combined with cell-sorting, opening the way for several applications that could expand the repertoire of tools in the hands of the neuroscientist, ranging from establishment of primary cultures derived from pure cell populations, to gene-expression analyses and biochemical or functional assays on well-defined cell subtypes in the context of neurodegenerative diseases, upon pharmacological treatment or gene therapy.

We present a flow cytometry method to identify simultaneously different cell types retrieved from mouse brain or spinal cord. This method could be exploited to isolate or characterize pure cell populations in neurodegenerative diseases or to quantify the extent of cell targeting upon in vivo administration of viral vectors or nanoparticles.

Recent advances in viral vector and nanomaterial sciences have opened the way for new cutting-edge approaches to investigate or manipulate the central ...

Quality-Controlled Sputum Analysis by Flow Cytometry

Sputum, widely used to study the cellular content and other microenvironmental features to understand the health of the lung, is traditionally analyzed using cytology-based methodologies. Its utility is limited because reading the slides is time-consuming and requires highly specialized personnel. Moreover, extensive debris and the presence of too many squamous epithelial cells (SECs), or cheek cells, often renders a sample inadequate for diagnosis. In contrast, flow cytometry allows for high-throughput phenotyping of cellular populations while simultaneously excluding debris and SECs.
The protocol presented here describes an efficient method to dissociate sputum into a single cell suspension, antibody stain and fix cellular populations, and acquire samples on a flow cytometric platform. A gating strategy that describes the exclusion of debris, dead cells (including SECs) and cell doublets is presented here. Further, this work also explains how to analyze viable, single sputum cells based on a cluster of differentiation (CD)45 positive and negative populations to characterize hematopoietic and epithelial lineage subsets. A quality control measure is also provided by identifying lung-specific macrophages as evidence that a sample is derived from the lung and is not saliva. Finally, it has been demonstrated that this method can be applied to different cytometric platforms by providing sputum profiles from the same patient analyzed on three flow cytometers; Navios EX, LSR II, and Lyric. Furthermore, this protocol can be modified to include additional cellular markers of interest. A method to analyze an entire sputum sample on a flow cytometric platform is presented here that makes sputum amenable for developing high-throughput diagnostics of lung disease.

This protocol describes an efficient method for dissociating sputum into a single cell suspension and the subsequent characterization of cellular subsets on standard flow cytometric platforms.

Sputum, widely used to study the cellular content and other microenvironmental features to understand the health of the lung, is traditionally analyzed ...

Flow Cytometry and Fluorescence-Activated Cell Sorting (FACS): Isolation of Splenic B Lymphocytes

Source: Perchet Thibaut1,2,3, Meunier Sylvain1,2,3, Sophie Novault4, Rachel Golub1,2,3 
1 Unit for Lymphopoiesis, Department of Immunology, Pasteur Institute, Paris, France 
2 INSERM U1223, Paris, France 
3 Universit&#233; Paris Diderot, Sorbonne Paris Cit&#233;, Cellule Pasteur, Paris, France 
4 Flow Cytometry Platfrom, Cytometry and Biomarkers UtechS, Center for Translational Science, Pasteur Institute, Paris, France
The overall function of the immune system is to defend the body against infectious organisms and other invaders. White blood cells, or leukocytes, are the key players of the immune system. Upon infection, they are activated and initiate an immune response. Leukocytes can be divided into various sub-populations (e.g., myeloid cells, lymphocytes, dendritic cells) based on different parameters that can be biological, physical, and/or functional (e.g., size, granularity, and secretion). One way to characterize leukocytes is through their surface proteins, which are mainly receptors. Each leukocyte population expresses a specific combination of receptors (e.g., cytotoxic, activating, migration receptors) that can define subsets among populations. As the immune system encompasses a wide range of cell populations, it is essential to characterize them to decipher their participation in the immune response.
Flow cytometry (FC or FCM) is a widely used method for analyzing the expression of cell surface and intracellular molecules, characterizing and defining different cell types in a heterogenous cell mixture. Flow cytometers are composed of three main sub-systems: fluidics, optics, and electronics. The fluidics system transports the cells in a stream such that they pass in front of a laser one by one. The optics system consists of light sources (lasers) to illuminate the particles, optical filters to direct the resulting light, and fluorescent signals to appropriate detectors. Finally, the electronics system converts the detected light signals into electronic signals that can be processed by the computer. As an individual cell passes in front of the laser beam, it scatters light. A detector in front of the beam measures forward scatter (FS) and several detectors to the side measure side scatter (SC). The FS correlates with cell size and SC is proportional to the granularity of the cells. In this manner, cell populations can often be distinguished based on differences in their size and granularity alone.
In addition to analyzing a cell&#39;s size, shape, and complexity, flow cytometry is widely used for detecting the expression of cell surface receptors (1). This is accomplished by using fluorochrome-labelled monoclonal antibodies which bind to known cell-specific receptors. Upon excitation, these bound fluorochromes emit a light of specific wavelength, called emission wavelength, which can be detected and scored. Fluorescence measurements provide quantitative and qualitative data about fluorochrome-labeled cell surface receptors. Hematologists were first to use FC for the therapeutic follow up of immune cell populations (2). Now, it is used for a wide range of applications such as immunophenotyping, cell viability, gene expression, cell counting, and GFP analysis.
FACS (Fluorescent Activated Cell Sorter) is a specialized type of flow cytometry, which sorts a population of cells into subpopulation using fluorescent labelling. Just like conventional flow cytometry, first FS, SC, and fluorescent data are collected. Then, the machine applies a charge (negative or positive) and an electrostatic deflection system (electromagnets) facilitates the collection of charged droplets containing cells into appropriate tubes.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/10494/10494fig1.jpg" /> 
Figure 1: Schematic representation of FACS. Sample (1) is aspirated in the FACS (2) and passed in front of the laser (3). Cell fluorescence is sensed by fluorescence detectors (4). Finally, cells are incorporated in droplets and the cells of interest are deflected by deflection plates (5) and collected in a collection tube (6). The remaining cells go into the trash (7). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/10494/10494fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The sorting aspect of the FACS presents many advantages. Many tests can help understand the role of specific cells in immune system, such as analyses of gene expression like RT-qPCR, cell cycle, or cytokine secretion. However, cells should be purified upstream to obtain clear and specific results. Here, FACS comes in useful and the desired cells can be sorted with great purity, yielding highly reliable and reproducible results. FACS can also be used to sort cells based on nuclear or other intracellular staining and according to the presence, absence, and density of surface receptors. FACS is now a standard technique for the purification of subpopulations of cells and has the ability sort up to four populations simultaneously.
This lab exercise demonstrates how to isolate splenic leukocytes and then how to specifically sort B lymphoid cells from the splenic leukocyte cell mixture using FACS.

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

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