R01AI132419, CU | AMC ImmunoMicro Flow Cytometry Shared Resource, RRID:SCR_021321, Many thanks to our colleagues at Cytek for continual discussions of full spectral cytometric analysis on the Aurora and SpectroFlo software. Figures were created with BioRender.com.

Mice were maintained at the University of Colorado Anschutz Medical campus in accordance with IACUC protocols. All mice were euthanized according to AAALAC standards.
1. Preparation of immune cells from the mouse spleen
NOTE: Euthanize na&#239;ve mice with CO2 euthanasia. C57BL/6 mice purchased from Jackson Laboratories and bred in-house are used for experiments at 6-12 weeks of age. Both male and female mice are utilized for experiments.
<ol>
	<li>Disinfect the mouse skin with 70% ethanol in order to reduce the possibility of external contaminants from getting into the sample.</li>
	<li>Dissect the mouse spleen using dissection tools, surgical scissors, and forceps. Harvest the spleen and place the detached spleen into 50 mL conical tube with ~5 mL of complete RPMI media (cRPMI) on ice. 
	NOTE: Complete RPMI is comprised of 10% FBS, 2 mM L-glutamine, and 1% penicillin/streptomycin.
	<ol>
		<li>To harvest the mouse spleen, make a 5 cm incision into the fur and skin along the left side of the mouse halfway between the front and back legs with scissors. Open the body cavity ~5 cm and remove the spleen using forceps. The spleen is the color of a kidney bean and is longer and flatter than the neighboring kidney.</li>
	</ol>
	</li>
	<li>Pour the contents of step 1.2 onto a sterile 70 &#181;m filter attached to a new 50 mL conical tube. Mechanically separate the spleen into a single cell suspension by pushing the spleen through the filter with a 5 mL syringe plunger until no spleen pulp remains on the filter surface.</li>
	<li>Pellet the splenocytes utilizing a swing-out rotor countertop centrifuge at 500 x g for 5 min at 4 &#730;C.</li>
	<li>Carefully decant the supernatant. The pelleted splenocytes remain at the bottom of the 50 mL conical tube.</li>
	<li>To remove red blood cells, re-suspend the pelleted splenocytes in 1 mL of ACK lysis buffer for 3 min according to the manufacturer&#39;s instructions. Mix well.</li>
	<li>Quench the ACK lysis buffer with 15 mL of cRPMI.</li>
	<li>Pellet the lymphocytes as directed in step 1.4.</li>
	<li>Re-suspend the cell suspension in 5 mL of cRPMI in a 50 mL conical tube. Place the samples on ice.
	<ol>
		<li>To count the cells, transfer 10 &#181;L of the cell suspension to a 0.6 mL microcentrifuge tube and dilute at a 1:1 ratio with Trypan blue solution. Then, transfer to a hemocytometer or automated cell counting system.</li>
	</ol>
	</li>
	<li>After counting, depending on the cell concentration in cell suspension as in step 1.9, pellet the cells in a tabletop centrifuge at 500 x g at 4 &#730;C for 5 min to prepare for the addition of cRMPI media containing Indo-1 AM ester dye.</li>
	<li>Calculate the volume for re-suspension of the cells to achieve 10-12 x 106 cells/mL. This is a 2x concentration of the total cell count required.</li>
	<li>Re-suspend the cells in the volume of cRPMI calculated in step 1.11. Place the cells at 37 &#730;C with 5% CO2 in either a 50 mL conical tube with the lid vented or a tissue culture plate.</li>
</ol>
2. Indo-1 ratiometric dye and fluorescent antibody labeling
<ol>
	<li>As per the manufacturer&#39;s instructions, add 50 &#181;L of DMSO into a vial containing 50 &#181;g of Indo-1 AM. The concentration of the stock solution is 1 &#181;g/&#181;L. 
	NOTE: DMSO is provided in micro-vials with the kit to prevent any oxidation of DMSO.</li>
	<li>Dilute the stock aliquot (1 &#181;g/&#181;L) into the appropriate experimental volume (established in 1.11) of cRPMI at a concentration of 6 &#181;g/mL, a 2x concentration. Do not store Indo-1 in an aqueous solution. 
	NOTE: Other buffers with added calcium can be used depending on the cell needs.</li>
	<li>Set complete media with Indo-1 aside in a water bath at 37 &#730;C. 
	NOTE: Use the minimum concentration of Indo-1 AM ester necessary to obtain an adequate signal. This needs to be titrated for varying cell types. Typically, a concentration between 3-5 &#181;M is sufficient. For primary T-cells, loading cells with Indo-1 at a concentration &#62;5 &#181;g/mL increases mean fluorescent intensity (MFI) above a log of 6 on spectral flow cytometers. If this occurs, decrease UV laser intensity and titrate the Indo-1 to a lower concentration.</li>
	<li>Dilute previously prepared cell suspension (in step 1.12), 1:1 in cRPMI, including the 2x Indo-1 media made in step 2.2. Ensure that the cells are at a concentration between 5-6 x 106/mL. 
	NOTE: Do not dye load unstained single stain control or surface marker single stain cell controls with Indo-1. Adding Indo-1 to single stain antibody controls will create a multi-color sample due to the Indo-1 added to single stained cells. Include indo-1 (calcium-free) EGTA added control to the sample plate created in step 2.6.</li>
	<li>Add 1 mL of cells/well/experimental condition into a 12-well tissue culture plate.</li>
	<li>Create a single stained control for Indo-1 (calcium-free) sample using previously Indo-1 dye-loaded cells and add EGTA to a final concentration of 2 mM. EGTA will chelate calcium in the cell suspension, giving a cleaner control sample.
	<ol>
		<li>Create an Indo-1 positive control (Indo-1 calcium-bound) by adding 50 &#181;M of ionomycin to the dye loaded cell suspension at the flow cytometer. Ionomycin is a membrane permeable calcium ionophore that binds calcium ions and facilitates the transfer of calcium ions into cells.</li>
	</ol>
	</li>
	<li>Create a well for biological negative control with no fluorophores or Indo-1 dye.</li>
	<li>Create a well for single stain controls for any additional cell populations of interest (antibodies user defined) using antibody conjugated fluorophores in a flow cytometry panel design. These will not include the Indo-1 ratiometric dye.</li>
	<li>Add the Fc receptor blocking antibody (~2 &#181;g/1 x 106 cells), as per the manufacturer&#39;s instructions, in advance of adding additional fluorochrome-conjugated antibodies for surface staining.</li>
	<li>Incubate the plate for 45 min at 37 &#730;C with 5% CO2.</li>
	<li>Resuspend the cells in a 12-well plate by gently tapping the plate every 15 min.</li>
	<li>Mix and remove the entire volume of cells suspended in the media from the 12-well plate. Then add the cell suspension to 1.7 mL microcentrifuge tubes.</li>
	<li>Pellet the cells in a microcentrifuge at 500 x g for 5 min at room temperature. Decant the supernatant and wash the cells by adding 1 mL of pre-warmed cRPMI. Pellet again and decant the supernatant. 
	NOTE: Washing the cells removes any remaining FC blocking antibody.</li>
	<li>Resuspend the cells in 1 mL of cRPMI in 1.7 mL microcentrifuge tubes and incubate each tube at 37 &#730;C for an additional 30 min at 5% CO2, leaving the top of the tube slightly open to allow for gas exchange. This allows complete de-esterification of the intracellular AM esters.</li>
	<li>Transfer the cells back into a 12-well tissue culture plate, if needed. Additional user defined titrated fluorochrome-conjugated antibodies can be added at a resting phase. 
	NOTE: Markers used in the methods panel include: Ghost 540, CD4, CD8, TCR&#946;, TCR&#947;&#948;, CD25, and CD1d/&#945;-galcer tetramer. Markers are chosen to analyze T-cell subsets.
	<ol>
		<li>Create a master mix of all the user-defined fluorochrome-conjugated antibodies according to the cell types of interest at a previously titrated volume into a 1.7 mL microcentrifuge tube.</li>
		<li>Add a calculated master mix for 1 mL of the cell volume, dependent on titrated antibodies to resting cells. 
		NOTE: The advantage of staining at step 2.15 instead of the later step 2.18 is that staining at step 2.15 will decrease the overall incubation time for the experiment. However, staining in a total volume of 1 mL at step 2.15 increases antibody usage.</li>
	</ol>
	</li>
	<li>After rest, pellet the cells at 500 x g for 5 min at room temperature. 
	NOTE: Cells in a 12-well tissue culture plate can be pelleted in the plate with a plate centrifuge accessory. Otherwise, pellet the cells in a 1.7 mL microcentrifuge tube.</li>
	<li>Wash the cells by resuspending the pellet in 1 mL of 4 &#730;C cold cRPMI and pellet the cells again as in step 2.16. Place the cells on ice. 
	NOTE: If surface staining cells separately, post de-esterification at step 2.18, less antibody volume is needed. Surface staining at this step can be performed after the rest phase in cold flow buffer (1x DPBS with added 1% FBS), using user-defined antibodies, on ice for 30 min. Wash the cells after the stain in cRPMI as in step 2.17.
	<ol>
		<li>Add viability stain Ghost540 diluted 1:7,500 in DPBS to samples according to the manufacturer&#39;s instructions. Heat kill the cells at 55 &#730;C on a warming block for single stained live/dead control. 
		NOTE: Viability functional dye staining to eliminate dead cells in flow cytometric analysis is performed without FBS. FBS can interact with amine dyes as per manufacturer&#39;s instructions.</li>
	</ol>
	</li>
	<li>Re-suspend individual samples in 500 &#181;L of cRPMI (phenol red-free) using a 5 mL polystyrene flow tube with a cap. 
	NOTE: Cells can be left at 4 &#730;C for up to an hour.</li>
	<li>Warm every 5 mL tube containing cells, individually, to 37 &#730;C using a bead bath for 7 min prior to the analysis on the flow cytometer keeping time consistent between the samples. Use a timer.</li>
</ol>
3. Bead bath tube: maintaining temperature during calcium flux analysis
<ol>
	<li>Add bath beads to a small water bath, with no water added; warm up to 37 &#730;C.</li>
	<li>Carefully cut a 50 mL conical tube in half, at the 25 mL mark, with a razor blade; discard the top of the tube.</li>
	<li>Fill the halved tube &#190; of the way with bath beads.</li>
	<li>Place the tube created in step 3.2 into the bead bath created in step 3.1. Bring the tube and beads to 37 &#730;C.</li>
	<li>Plug the bead bath into an electrical outlet next to the flow cytometer in order to maintain the temperature in preparation for sample analysis.</li>
	<li>Add a 50 mL conical Styrofoam rack cut to hold one tube to position the tube in place while running on the flow cytometer. This will eliminate the need to manually hold the tube for the duration of the curve analysis.</li>
</ol>
4. Acquisition of calcium influx using flow cytometric analysis
<ol>
	<li>Log into the flow cytometer software on a flow analyzer.</li>
	<li>Click on the Library tab to add Indo-1 (calcium-bound) and Indo-1 (calcium-free) fluorescent tags. Select Fluorescent Tags on the software menu on the left. Select UV laser under fluorescent tag groups and click on +add to add a fluorescent tag name (Indo-1 (calcium-bound)/Indo-1 (calcium-free)) to the library. Choose the laser excitation wavelength and the emission wavelength, and then click on Save. Continue to the experimental setup. 
	NOTE: Indo-1 is excited by the UV laser at a wavelength of 355 nm. The emission wavelength of Indo-1 (calcium-bound) is 372 nm. The emission wavelength of Indo-1 (calcium-free) is 514 nm.</li>
	<li>Click on the Acquisition tab. Open/create a new experiment and add the desired fluorescent tags to the experiment.</li>
	<li>Create Reference Group and a new sample group/s for the experiment. Label both the samples and the reference groups as necessary.</li>
	<li>Under the Acquisitions tab, set the events to record to the maximum: 10,000,000.</li>
	<li>Set the stopping volume to the maximum volume: 3,000 &#181;L.</li>
	<li>Set the stopping time to the maximum time: 36,000 s.</li>
	<li>Acquire single stained controls for defined conjugated-antibodies included in the multicolor panel.</li>
	<li>Acquire single stained controls for Indo-1 functional dye.
	<ol>
		<li>Add 50 &#181;M of ionomycin to the calcium bound Indo-1 positive control. Vortex to mix. Add the flow tube to the sample injection port and click on Record.</li>
		<li>Add calcium free Indo-1 negative control to the flow cytometer and click on Record. EGTA was added in step 2.6.</li>
		<li>Unmix the single stained controls by clicking on the Unmix button. 
		NOTE: All the single stained controls from all the conjugated antibodies and functional dyes must be recorded prior to unmixing the samples. For the Unmix button to function, all the reference controls need to be recorded first. Unmixing can be done before or after sample collection.</li>
	</ol>
	</li>
	<li>Once unmixing is complete, create sequential plots using the unmixed worksheet. SSC-A versus FSC-A removing debris from sample, SSC-A versus SSC-H to remove doublets. This is followed by a viability clean-up gate along with further gating on populations of interest. To create gates, click on Plot. Add a plot to the worksheet, and double click inside the gate to create a downstream gated population. Continue until all the populations of interest are accounted for.</li>
	<li>Warm single stained control samples (from steps 2.6-2.9 in section Indo-1 ratiometric dye and fluorescent antibody labeling) to 37 &#730;C&#160;for sequential analysis. Set up the flow cytometry software.</li>
	<li>Run DI water on the cytometer for 2-3 min to ensure the fluidic stability of the flow cytometer.</li>
	<li>Set up sequential plots on the unmixed worksheet by creating gates using the Polygon, Rectangle or Ellipse gate buttons. Gate to include the population of interest (i.e., lymphocytes using SSC-A vs. FSC-A [size/lymphocytes discrimination]). Negative populations appear clustered around zero whereas positive populations can be visualized by increasing MFI. Populations of interest will be user defined based on the biological question.</li>
	<li>Double click inside the gate created and change Y-axis and X-axis parameters to SSC-A versus SSC-H (singlet discrimination). Complete defining the axis parameters by left clicking on the words on the axis.</li>
	<li>Create a plot with SSC-A versus viability dye signal (viability gate parameters). Gate on live cells, which are negative for amine dye staining. Live cells, negative for live dead staining, will cluster at the 0 mark on both the Y and X axes.</li>
	<li>Double click on the inside plot to create a new plot containing only single cell live lymphocytes. Change the Y-axis to Indo-1 (bound-calcium) (V1) and/or Indo-1 (free-calcium) (V7) versus time on the X-axis for visualization of the calcium influx.</li>
	<li>Place the warmed biological sample onto the machine SIT and run the samples at 2,500-3,000 events/s on medium to allow visualization of calcium influx in limited cell number populations (e.g., iNKT). 
	NOTE: By resuspending the cells at a concentration of 1 x 106/mL, the cell events collected at medium flow rate should equal 2,500-3,000 events/s. Lower the flow rate under acquisition control by clicking on the drop-down menu or dilute the sample if the cell suspension has an increased concentration. Running the samples at a high flow rate can increase the spreading of the signal from one fluorophore to other fluorophores in the panel.</li>
	<li>Add the next tube to the bead bath to warm to 37 &#730;C.</li>
	<li>In the Acquisition Control, press Record to record the initial data for 30 s to obtain a basal level of calcium signaling in the sample.</li>
	<li>In the Acquisition Control, click on Stop and remove the tube from Sample Injection Port (SIP).</li>
	<li>Add 30 &#181;g of unconjugated anti-CD3 directly into the sample tube to initiate calcium influx. The final concentration per tube is 60 &#181;g/mL. 
	NOTE: There is no washing step after the addition of anti-CD3.</li>
	<li>Place the tube back on machine SIP as quickly as possible and press Record in the software.</li>
	<li>Record data for 7 min, watching the time elapsed&#160;under the acquisition controls in the software, to observe the time-course of calcium influx within the sample populations.</li>
	<li>After 7 min, press Stop in the software and add 1 &#181;g/mL of ionomycin. Record for 30 s to obtain the maximum calcium signal in the cells of interest. To limit carryover of ionomycin into the next sample, complete two SIT flushes on the instrument between samples.</li>
	<li>Continue to the next sample and repeat the workflow until all the samples have been collected.</li>
	<li>Save the experiment and click on the export zip file. Unmixed files (.fsc) are uploaded to external flow cytometry analysis software.</li>
</ol>
5. Calcium curve analysis
<ol>
	<li>Using an analysis software6 for flow cytometry, convert Indo-1 fluorescent signals to a ratio for time-lapse calcium measurements. Derive parameters under the tools tab by inserting references Indo-1 (calcium-bound)/Indo-1 (free-calcium) using a linear scale. Set the minimum to zero and the maximum according to the influx of calcium ratio, typically around 10. The maximum ratio varies by cell type and MFI of Indo-1.</li>
	<li>Highlight the selected parameter. Under tools, add kinetic analysis to choose the parameter by clicking on the Kinetics tab. Set the Y-axis to derived.</li>
	<li>Select MFI (mean fluorescent intensity) within the Kinetics tab.</li>
	<li>Add Gaussian smoothing, if desired. To add Gaussian smoothing, select the option in the pull-down statistics menu in the flow analysis software. Gaussian smoothing can be added to smooth the visualization of the calcium influx curve of analysis.</li>
	<li>Create multiple ranges for statistics along the calcium flux time course for biological comparisons within the samples.</li>
	<li>Drag the samples into the layout editor and add the statistics as desired. Statistical analysis varies depending on the biological question. For publication purposes, multiple replicates are analyzed using t-test or ANOVA.</li>
	<li>For a moment of time analysis, set the gate on a specific moment in time along the calcium flux. Examine the desired surface markers and gate on the population of interest; then, assess a two-dimensional dot-plot of Indo-1 (calcium-free) versus Indo-1 (calcium bound) for gated cells within that moment in the time region.</li>
</ol>

<ol>
	<li>Weiss, A., Imboden, J., Shoback, D., Stobo, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+T3+surface+molecules+in+human+T-cell+activation:+T3-dependent+activation+results+in+an+increase+in+cytoplasmic+free+calcium.">Role of T3 surface molecules in human T-cell activation: T3-dependent activation results in an increase in cytoplasmic free calcium.</a> Proceedings of the National Academy of Sciences of the United States of America. 81 (13), 4169-4173 (1984).</li><li>Huang, G. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+calcium+mobilization+study+(flow+cytometry).">Cell calcium mobilization study (flow cytometry).</a> Bio-Protocol. 2 (9), 171 (2012).</li><li>June, C. H., Moore, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+intracellular+ions+by+flow+cytometry.">Measurement of intracellular ions by flow cytometry.</a> Current Protocols in Immunology. , (2004).</li><li>Posey, A. D., Kawalekar, O. U., June, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+intracellular+ions+by+flow+cytometry.">Measurement of intracellular ions by flow cytometry.</a> Current Protocols in Cytometry. 72, 1-21 (2015).</li><li>Grynkiewicz, G., Poenie, M., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+generation+of+Ca2++indicators+with+greatly+improved+fluorescence+properties.">A new generation of Ca2+ indicators with greatly improved fluorescence properties.</a> The Journal of Biological Chemistry. 260 (6), 3440-3450 (1985).</li><li>McKinnon, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry:+An+overview.">Flow cytometry: An overview.</a> Current Protocols in Immunology. 120, 1-11 (2018).</li><li>Nolan, J. P., Condello, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectral+flow+cytometry.">Spectral flow cytometry.</a> Current Protocols in Cytometry. , (2013).</li><li>Bonilla, D. L., Reinin, G., Chua, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Full+spectrum+flow+cytometry+as+a+powerful+technology+for+cancer+immunotherapy+research.">Full spectrum flow cytometry as a powerful technology for cancer immunotherapy research.</a> Frontiers in Molecular Biosciences. 7, 612801 (2021).</li><li>Zhong, Y., 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=Targeting+Interleukin-2-inducible+T-cell+Kinase+(ITK)+and+Resting+Lymphocyte+Kinase+(RLK)+using+a+novel+covalent+inhibitor+PRN694.">Targeting Interleukin-2-inducible T-cell Kinase (ITK) and Resting Lymphocyte Kinase (RLK) using a novel covalent inhibitor PRN694.</a> The Journal of Biological Chemistry. 290 (10), 5960-5978 (2015).</li><li>Gallagher, M. 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=Hierarchy+of+signaling+thresholds+downstream+of+the+T-cell+receptor+and+the+Tec+kinase+ITK.">Hierarchy of signaling thresholds downstream of the T-cell receptor and the Tec kinase ITK.</a> Proceedings of the National Academy of Sciences of the United States of America. 118 (35), 2025825118 (2021).</li><li>Andreotti, A. H., Schwartzberg, P. L., Joseph, R. E., Berg, L. J. <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+signaling+regulated+by+the+tec+family+kinase,+Itk.">T-Cell signaling regulated by the tec family kinase, Itk.</a> Cold Spring Harbor Perspectives in Biology. 2 (7), 002287 (2010).</li><li>Nakamura, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EGTA+can+inhibit+vesicular+release+in+the+nanodomain+of+single+Ca2++channels.">EGTA can inhibit vesicular release in the nanodomain of single Ca2+ channels.</a> Frontiers in Synaptic Neurosciene. 11, 26 (2019).</li><li>Cytek Aurora Users Guide. Cytek Available from: <a target="_blank" href="https://depts.washington.edu/flowlab/Cell%20Analysis%20Facility/Aurora%20User%20Guide.pdf">https://depts.washington.edu/flowlab/Cell%20Analysis%20Facility/Aurora%20User%20Guide.pdf</a> (2022)</li><li>SpctroFlo Software. Cytek Available from: <a target="_blank" href="https://cytekbio.com/pages/spectro-flo">https://cytekbio.com/pages/spectro-flo</a> (2022)</li><li>FlowJo Software. Becton-Dickinson Available from: <a target="_blank" href="https://www.flowjo.com/solutions/flowjo">https://www.flowjo.com/solutions/flowjo</a> (2022)</li><li>Godfrey, D. I., Zlotnik, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+points+in+early+T-cell+development.">Control points in early T-cell development.</a> Immunology Today. 14 (11), 547-553 (1993).</li><li>Vossen, A. 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The authors have no competing interests to declare.

This protocol describes an optimized assay designed to measure calcium responses in primary murine T-cells loaded with titrated Indo-1 ratiometric indicator dye using full spectrum flow cytometry7,8. The advantage of performing calcium flux assays using full spectrum flow cytometry is the ability to multiplex surface cell marker staining in combination with assessment of Indo-1 fluorescence. Full spectrum flow cytometry has the advantage of allowing the use of hi...

T-cell receptor (TCR) induced calcium influx is a useful measure of T-cell activation and is frequently used to determine whether a population of T-cells has impaired responses in the proximal steps of the TCR signaling pathway1. Measurements of calcium influx are generally performed by pre-labeling the T-cells with one or a pair of fluorescent calcium indicator dyes, and then examining the fluorescent signals using flow cytometry in real-time after TCR cross-linking2,3,4. Indo-1, a ratio-metric calcium dye, is excited by the UV laser with peak emissions at two different wavelengths dependent on calcium binding5, and is a commonly used indicator dye for flow cytometry analysis of calcium responses in live lymphocytes. As the emission profile of Indo-1 is quite broad, it can be challenging to combine Indo-1 assessment with simultaneous analysis of multiple cell surface markers by band-pass flow cytometry. This limitation restricts the utilization of flow cytometry analysis of calcium responses to pre-purified populations of T-cells or to populations identified by a limited set of cell surface molecules.
To address the limitations of measuring calcium responses on heterogenous populations of primary lymphocytes using band-pass flow cytometry, a protocol was developed to measure Indo-1 fluorescence using full spectrum flow cytometry. This method allows for multiplexing Indo-1 with panels of antibodies directed at cell surface molecules, taking advantage of the highly flexible capabilities of full spectrum flow cytometry. The advantage of using full spectrum flow cytometry over conventional flow cytometry is its ability to distinguish the fluorescent signals from highly overlapping dyes, thereby increasing the number of surface markers that can be simultaneously assessed in each sample. Conventional flow cytometry uses bandpass filters and is restricted to one fluorochrome per detector system6. Full spectrum flow cytometry collects signals across the entire spectrum of the fluorochrome using 64 detectors on a five-laser spectral flow cytometry system7,8. In addition, full spectrum flow cytometry takes advantage of APD (Avalanche Photo Diode) detectors that have increased sensitivity relative to photomultiplier tube detectors present on conventional flow cytometers8. Consequently, this approach is ideal for the heterogeneous cell populations, such as peripheral blood mononuclear cells or murine secondary lymphoid organ cell suspensions, as it eliminates the need for the isolation of specific T-cell populations prior to calcium dye labeling. Instead, cell surface marker expression profiles and flow cytometry gating after data collection can be used to assess calcium responses in each population of interest. As shown in this report, Indo-1 can readily be combined with eight fluorochrome-conjugated antibodies, resulting in a total of 10 unique spectral signatures. Furthermore, this method can be readily applied to mixtures of cells from congenically distinct mouse lines, allowing for the simultaneous analysis of calcium responses in wild-type T-cells compared to those from a gene-targeted mouse line.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>12 well TC treated plates</td>
			<td>Cell Treat</td>
			<td>229111</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL conical</td>
			<td>Greiner Bio1</td>
			<td>41-12-17-03</td>
			<td>50 mL Polypropylene centrifuge tubes with cap</td>
		</tr>
		<tr>
			<td>5mL polysterene flow tubes</td>
			<td>Corning</td>
			<td>352052</td>
			<td></td>
		</tr>
		<tr>
			<td>5mL syringe</td>
			<td>BD syringe</td>
			<td>309646</td>
			<td>plunger only is used sheith is discarded</td>
		</tr>
		<tr>
			<td>70uM filter</td>
			<td>Greiner bio1</td>
			<td>542070</td>
			<td></td>
		</tr>
		<tr>
			<td>aCD3 (17A2)</td>
			<td>Biolegend</td>
			<td>100202</td>
			<td></td>
		</tr>
		<tr>
			<td>AKC lysis Buffer</td>
			<td>Gibco</td>
			<td>A1049201</td>
			<td></td>
		</tr>
		<tr>
			<td>Aurora Spectral Flowcytometer</td>
			<td>https://cytekbio.com/pages/aurora</td>
			<td></td>
			<td></td>
		</tr>
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			<td>Bath Beads</td>
			<td>coleparmer</td>
			<td>Item # UX-06274-52</td>
			<td></td>
		</tr>
		<tr>
			<td>CD19 PE</td>
			<td>Tonbo</td>
			<td>50-0193-U100</td>
			<td></td>
		</tr>
		<tr>
			<td>CD1d Tetramer APC</td>
			<td>NIH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CD25 PECy7</td>
			<td>ebioscience</td>
			<td>15-0251</td>
			<td></td>
		</tr>
		<tr>
			<td>CD4 APC Cy7</td>
			<td>Tonbo</td>
			<td>25-0042-U100</td>
			<td></td>
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		<tr>
			<td>CD8a FITC</td>
			<td>ebioscience</td>
			<td>11-0081-85</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Incubator</td>
			<td>Formal Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection Tools forceps</td>
			<td>McKesson</td>
			<td>#487593</td>
			<td>Tissue Forceps McKesson Adson 4-3/4 Inch Length Office Grade Stainless Steel NonSterile NonLocking Thumb Handle 1 X 2 Teeth</td>
		</tr>
		<tr>
			<td>Dissection Tools Scissors</td>
			<td>McKesson</td>
			<td>#970135</td>
			<td>Operating Scissors McKesson Argent&#8482; 4-1/2 Inch Surgical Grade Stainless Steel Finger Ring Handle Straight Sharp Tip / Sharp Tip</td>
		</tr>
		<tr>
			<td>DPBS 1x</td>
			<td>Gibco</td>
			<td>14190-136</td>
			<td>DPBS&#160;</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Fisher</td>
			<td>NC1280093</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Hyclond</td>
			<td>SH30071.03</td>
			<td>lot AE29165301</td>
		</tr>
		<tr>
			<td>FlowJo Software</td>
			<td>https://www.flowjo.com/</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Indo1-AM Ester Dye</td>
			<td>ebioscience</td>
			<td>65-085-39</td>
			<td>Calcium Loading Dye&#160;</td>
		</tr>
		<tr>
			<td>ionomycin</td>
			<td>Millipore</td>
			<td>407951-1mg</td>
			<td></td>
		</tr>
		<tr>
			<td>Live/Dead Ghost 540</td>
			<td>Tonbo</td>
			<td>13-0879-T100</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes 1.7mL</td>
			<td>Light Labs</td>
			<td>A-7001</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin/L-Glutamine</td>
			<td>Gibco</td>
			<td>10378-016</td>
			<td></td>
		</tr>
		<tr>
			<td>PRN694</td>
			<td>Med Chem Express</td>
			<td>Hy-12688</td>
			<td></td>
		</tr>
		<tr>
			<td>Purified Anti-Mouse CD16/CD32 (FC Shield) (2.4G2)</td>
			<td>Tonbo</td>
			<td>70-0161-M001</td>
			<td>FC Block</td>
		</tr>
		<tr>
			<td>RPMI</td>
			<td>Gibco</td>
			<td>1875093</td>
			<td>&#160;+ phenol red</td>
		</tr>
		<tr>
			<td>RPMI phenol free</td>
			<td>Gibco</td>
			<td>11835030</td>
			<td>&#160;-phenol red</td>
		</tr>
		<tr>
			<td>Table top centrifuge</td>
			<td>Beckman Coulter</td>
			<td>Allegra612</td>
			<td></td>
		</tr>
		<tr>
			<td>TCR&#946; PerCP Cy5.5</td>
			<td>ebioscience</td>
			<td>45-5961-82</td>
			<td></td>
		</tr>
		<tr>
			<td>TCR&#947;/&#948; Pe Cy5</td>
			<td>ebioscience</td>
			<td>15-5961-82</td>
			<td></td>
		</tr>
		<tr>
			<td>Vi-Cell Blu Reagent Pack</td>
			<td></td>
			<td>Product No: C06019</td>
			<td>Includes Tripan</td>
		</tr>
		<tr>
			<td>Vi-Cell Blu</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Waterbath</td>
			<td>Fisher Brand Dry bath</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

analysis of t-cell receptor-induced calcium influx in primary murine t-cells by full spectrum flow cytometry

Calcium influx in response to T-cell receptor stimulation is a common measure of T-cell signaling. Several calcium indicator dyes have been developed to assess calcium signaling by band-pass flow cytometry. This protocol is designed to measure calcium responses in primary murine T-cells using full spectrum flow cytometry. Total splenocytes are labeled with the ratiometric calcium indicator dye Indo-1, along with a panel of fluorochrome-conjugated antibodies to cell surface molecules. Leveraging the capabilities of full spectrum flow cytometry provides a platform for utilizing a wide array of cell surface stains in combination with Indo-1. Cells are then analyzed in real-time at 37 &#176;C before and after the addition of an anti-CD3 antibody to stimulate the T-cell receptor. After unmixing the spectral signals, the ratio of calcium-bound to calcium-free Indo-1 is calculated and can be visualized over time for each gated population of splenocytes. This technique can allow for the simultaneous analysis of calcium responses in multiple cell populations.

The experimental workflow to assess calcium responses in primary murine T-cells using an Indo-1 ratiometric dye with multiplexing surface stains is shown in Figure 1. After harvesting and processing the mouse splenocytes into a single cell suspension, the cells are stained with an Indo-1 AM ester and surface stained with fluorochrome-linked antibodies. Once the dye loading and antibody staining are completed, the lymphocyte samples are warmed up to a biological temperature (37 &#176;C). The ...

Watch this Scientific Journal Video about Analysis of T-cell Receptor-Induced Calcium Influx in Primary Murine T-cells by Full Spectrum Flow Cytometry at JoVE.com

Analysis of T-cell Receptor-Induced Calcium Influx in Primary Murine T-cells by Full Spectrum Flow Cytometry

Calcium influx, a measure of T-cell signaling, is an effective way to analyze responses to T-cell receptor stimulation. This protocol for multiplexing Indo-1 with panels of antibodies directed at cell surface molecules takes advantage of the highly flexible capabilities of full spectrum flow cytometry.

Calcium influx in response to T-cell receptor stimulation is a common measure of T-cell signaling. Several calcium indicator dyes have been developed to ...

analysis-t-cell-receptor-induced-calcium-influx-primary-murine-t

Research

JoVE Journal

Immunology and Infection

3.7K Views.  University of Colorado-Anschutz School of Medicine. Calcium influx, a measure of T-cell signaling, is an effective way to analyze responses to T-cell receptor stimulation. This protocol for multiplexing Indo-1 with panels of antibodies directed at cell surface molecules takes advantage of the highly flexible capabilities of full spectrum flow cytometry.

Video: Analysis of T-cell Receptor-Induced Calcium Influx in Primary Murine T-cells by Full Spectrum Flow Cytometry

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam cells, immune cells, vascular endothelial and smooth muscle cells, platelets, extracellular matrix, and a lipid-rich core with extensive necrosis and fibrosis of surrounding tissues.1 The innate and adaptive arms of the immune response are involved in the initiation, development and persistence of atherosclerosis.2, 3 There is a significant body of evidence that different subsets of the immune cells, such as macrophages, dendritic cells, T and B lymphocytes, are present within the aortas of healthy and atherosclerosis-prone mice4. Additionally, immune cells are found in the surrounding aortic adventitia which suggests an important role of this tissue in atherogenesis.2
For some time, the quantitative detection of different types of immune cells, their activation status, and the cellular composition within the aortic wall was limited by RT-PCR and immunohistochemical methods for the study of atherosclerosis. Few attempts were made to perform flow cytometry using human aortas, and a number of problems, such as a high autofluorescence, have been reported5,6. Human atherosclerotic plaques were digested with collagenase 1, and free cells were collected and stained for CD14+/CD11c+ to highlight macrophage-derived foam cells. In this study, a "mock" channel was used to avoid false-positive staining.6 Necrotic materials accumulating during the digestion process give rise in a large amount of debris that generates a high autofluorescence in aortic samples. To resolve this problem, a panel of negative and positive controls has been proposed, but only double staining could be applied in these samples. We have developed a new flow cytometry-based method7 to analyze the immune cell composition and characterize the activation, proliferation, differentiation of immune cells in healthy and atherosclerosis-prone aorta. This method allows the investigation of the immune cell composition of the aortic wall and opens possibilities to use a broad spectrum of immunological methods for investigations of immune aspects of this disease.

This paper presents a flow cytometry-based method to investigate the immune composition of aortas. The paper also illustrates an additional technique that allows examining surrounding adventitia and vessel wall separately. This method opens possibilities to perform phenotypical analyses of aortic leukocytes and apply several immunological assays for atherosclerosis studies.

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam ...

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

The Use of Flow Cytometry to Assess the State of Chromatin in T Cells

During a proper immune response, quiescent T cells become activated upon antigen presentation to their antigen-specific T cell receptor. This leads to clonal proliferation of only those T cells that bear a receptor that recognizes the antigen. Chromatin decondensation is a hallmark of T cell activation and is required for T cells to acquire the ability to proliferate after antigen engagement. This change in chromatin condensation can be detected using antibodies raised against histone proteins. These antibodies cannot bind to their epitopes in na&#239;ve T cells as well as they can in activated T cells. We describe how to simultaneously stain T cell-specific surface markers, track viability with a fixable dead cell stain, and measure chromatin status via intracellular staining of Histone H3 proteins. Stained cells are analyzed by flow cytometry and chromatin condensation status is measured as the mean fluorescence intensity (MFI) of the Histone H3 stain. Chromatin decondensation during T cell activation is demonstrated as an increase in the MFI

Flow cytometry can be utilized to assess the state of chromatin within T cells. This protocol allows scientists to interpret evidence of chromatin decondensation during T cell activation demonstrated by an increase in the mean florescence intensity (MFI) of fluorescent Histone H3 antibodies

During a proper immune response, quiescent T cells become activated upon antigen presentation to their antigen-specific T cell receptor. This leads to ...

Measurement of T Cell Alloreactivity Using Imaging Flow Cytometry

The measurement of immunological reactivity to donor antigens in transplant recipients is likely to be crucial for the successful reduction or withdrawal of immunosuppression. The mixed leukocyte reaction (MLR), limiting dilution assays, and trans-vivo delayed-type hypersensitivity (DTH) assay have all been applied to this question, but these methods have limited predictive ability and/or significant practical limitations that reduce their usefulness. Imaging flow cytometry is a technique that combines the multiparametric quantitative powers of flow cytometry with the imaging capabilities of fluorescent microscopy. We recently made use of an imaging flow cytometry approach to define the proportion of recipient T cells capable of forming mature immune synapses with donor antigen-presenting cells (APCs). Using a well-characterized mouse heart transplant model, we have shown that the frequency of in vitro immune synapses among T-APC membrane contact events strongly predicted allograft outcome in rejection, tolerance, and a situation where transplant survival depends on induced regulatory T cells. The frequency of T-APC contacts increased with T cells from mice during acute rejection and decreased with T cells from mice rendered unresponsive to alloantigen. The addition of regulatory T cells to the in vitro system reduced prolonged T-APC contacts. Critically, this effect was also seen with human polyclonally expanded, naturally occurring regulatory T cells, which are known to control the rejection of human tissues in humanized mouse models. Further development of this approach may allow for a deeper characterization of the alloreactive T-cell compartment in transplant recipients. In the future, further development and evaluation of this method using human cells may form the basis for assays used to select patients for immunosuppression minimization, and it can be used to measure the impact of tolerogenic therapies in the clinic.

This paper describes a method for measuring alloreactivity in a mixed population of T cells using imaging flow cytometry.

The measurement of immunological reactivity to donor antigens in transplant recipients is likely to be crucial for the successful reduction or withdrawal ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Multicolor Flow Cytometry-based Quantification of Mitochondria and Lysosomes in T Cells

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular components responsible for supplying cell energy; however, excess mitochondria also produce reactive oxygen species (ROS) that could cause cell death. Therefore, the number of mitochondria must constantly be adjusted to fit the needs of the cells. This dynamic regulation is achieved in part through the function of lysosomes that remove surplus/damaged organelles and macromolecules. Hence, cellular mitochondrial and lysosomal contents are key indicators to evaluate the metabolic adjustment of cells. With the development of probes for organelles, well-characterized lysosome or mitochondria-specific dyes have become available in various formats to label cellular lysosomes and mitochondria. Multicolor flow cytometry is a common tool to profile cell phenotypes, and has the capability to be integrated with other assays. Here, we present a detailed protocol of how to combine organelle-specific dyes with surface markers staining to measure the amount of lysosomes and mitochondria in different T cell populations on a flow cytometer.

This article illustrates a powerful method to quantify mitochondria or lysosomes in living cells. The combination of lysosome- or mitochondria-specific dyes with fluorescently conjugated antibodies against surface markers allows the quantification of these organelles in mixed cell populations, like primary cells harvested from tissue samples, by using multicolor flow cytometry.

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular ...

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, ...

Preparation of Whole Bone Marrow for Mass Cytometry Analysis of Neutrophil-lineage Cells

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a myeloid-biased 39-antibody CyTOF panel to evaluate the hematopoietic system with a focus on the neutrophil-lineage cells by using this protocol. The CyTOF result was analyzed with an open-resource dimensional reduction algorithm, viSNE, and the data was presented to demonstrate the outcome of this protocol. We have discovered new neutrophil-lineage cell populations based on this protocol. This protocol of fresh whole BM preparation may be used for 1), CyTOF analysis to discover unidentified cell populations from whole BM, 2), investigating whole BM defects for patients with blood disorders such as leukemia, 3), assisting optimization of fluorescence-activated flow cytometry protocols that utilize fresh whole BM.

Here, we present a protocol to process fresh bone marrow (BM) isolated from mouse or human for high-dimensional mass cytometry (Cytometry by Time-Of-Flight, CyTOF) analysis of neutrophil-lineage cells.

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a ...

Assessing the Expression of Major Histocompatibility Complex Class I on Primary Murine Hippocampal Neurons by Flow Cytometry

Increasing evidence supports the hypothesis that neuro-immune interactions impact nervous system function in both homeostatic and pathologic conditions. A well-studied function of major histocompatibility complex class I (MHCI) is the presentation of cell-derived peptides to the adaptive immune system, particularly in response to infection. More recently it has been shown that the expression of MHCI molecules on neurons can modulate activity-dependent changes in the synaptic connectivity during normal development and neurologic disorders. The importance of these functions to the brain health supports the need for a sensitive assay that readily detects MHCI expression on neurons. Here we describe a method for primary culture of murine hippocampal neurons and then assessment of MHCI expression by flow cytometric analysis. Murine hippocampus is microdissected from prenatal mouse pups at the embryonic day 18. Tissue is dissociated into a single cell suspension using enzymatic and mechanical techniques, then cultured in a serum-free media that limits growth of non-neuronal cells. After 7 days in vitro, MHCI expression is stimulated by treating cultured cells pharmacologically with beta interferon. MHCI molecules are labeled in situ with a fluorescently tagged antibody, then cells are non-enzymatically dissociated into a single cell suspension. To confirm the neuronal identity, cells are fixed with paraformaldehyde, permeabilized, and labeled with a fluorescently tagged antibody that recognizes neuronal nuclear antigen NeuN. MHCI expression is then quantified on neurons by flow cytometric analysis. Neuronal cultures can easily be manipulated by either genetic modifications or pharmacologic interventions to test specific hypotheses. With slight modifications, these methods can be used to culture other neuronal populations or to assess expression of other proteins of interest.

This protocol describes a method for culturing primary hippocampal neurons from embryonic mouse brain. The expression of major histocompatibility complex class I on the extracellular surface of cultured neurons is then assessed by flow cytometric analysis.

Increasing evidence supports the hypothesis that neuro-immune interactions impact nervous system function in both homeostatic and pathologic conditions. A ...