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

Podsumowanie

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.

Streszczenie

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 °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.

Wprowadzenie

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-linking^2,3,4. 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 system^7,8. 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.

Protokół

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 naïve 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.

1. Disinfect the mouse skin with 70% ethanol in order to reduce the possibility of external contaminants from getting into the sample.
2. 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.
   1. 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.
3. Pour the contents of step 1.2 onto a sterile 70 µm 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.
4. Pellet the splenocytes utilizing a swing-out rotor countertop centrifuge at 500 x g for 5 min at 4 ˚C.
5. Carefully decant the supernatant. The pelleted splenocytes remain at the bottom of the 50 mL conical tube.
6. To remove red blood cells, re-suspend the pelleted splenocytes in 1 mL of ACK lysis buffer for 3 min according to the manufacturer's instructions. Mix well.
7. Quench the ACK lysis buffer with 15 mL of cRPMI.
8. Pellet the lymphocytes as directed in step 1.4.
9. Re-suspend the cell suspension in 5 mL of cRPMI in a 50 mL conical tube. Place the samples on ice.
   1. To count the cells, transfer 10 µL 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.
10. 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 ˚C for 5 min to prepare for the addition of cRMPI media containing Indo-1 AM ester dye.
11. 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.
12. Re-suspend the cells in the volume of cRPMI calculated in step 1.11. Place the cells at 37 ˚C with 5% CO₂ in either a 50 mL conical tube with the lid vented or a tissue culture plate.

2. Indo-1 ratiometric dye and fluorescent antibody labeling

1. As per the manufacturer's instructions, add 50 µL of DMSO into a vial containing 50 µg of Indo-1 AM. The concentration of the stock solution is 1 µg/µL.
   NOTE: DMSO is provided in micro-vials with the kit to prevent any oxidation of DMSO.
2. Dilute the stock aliquot (1 µg/µL) into the appropriate experimental volume (established in 1.11) of cRPMI at a concentration of 6 µg/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.
3. Set complete media with Indo-1 aside in a water bath at 37 ˚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 µM is sufficient. For primary T-cells, loading cells with Indo-1 at a concentration >5 µg/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.
4. 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.
5. Add 1 mL of cells/well/experimental condition into a 12-well tissue culture plate.
6. 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.
   1. Create an Indo-1 positive control (Indo-1 calcium-bound) by adding 50 µM 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.
7. Create a well for biological negative control with no fluorophores or Indo-1 dye.
8. 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.
9. Add the Fc receptor blocking antibody (~2 µg/1 x 10⁶ cells), as per the manufacturer's instructions, in advance of adding additional fluorochrome-conjugated antibodies for surface staining.
10. Incubate the plate for 45 min at 37 ˚C with 5% CO_2.
11. Resuspend the cells in a 12-well plate by gently tapping the plate every 15 min.
12. 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.
13. 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.
14. Resuspend the cells in 1 mL of cRPMI in 1.7 mL microcentrifuge tubes and incubate each tube at 37 ˚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.
15. 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, TCRβ, TCRγδ, CD25, and CD1d/α-galcer tetramer. Markers are chosen to analyze T-cell subsets.
    1. 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.
    2. 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.
16. 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.
17. Wash the cells by resuspending the pellet in 1 mL of 4 ˚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.
    1. Add viability stain Ghost540 diluted 1:7,500 in DPBS to samples according to the manufacturer's instructions. Heat kill the cells at 55 ˚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's instructions.
18. Re-suspend individual samples in 500 µL of cRPMI (phenol red-free) using a 5 mL polystyrene flow tube with a cap.
    NOTE: Cells can be left at 4 ˚C for up to an hour.
19. Warm every 5 mL tube containing cells, individually, to 37 ˚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.

3. Bead bath tube: maintaining temperature during calcium flux analysis

1. Add bath beads to a small water bath, with no water added; warm up to 37 ˚C.
2. Carefully cut a 50 mL conical tube in half, at the 25 mL mark, with a razor blade; discard the top of the tube.
3. Fill the halved tube ¾ of the way with bath beads.
4. Place the tube created in step 3.2 into the bead bath created in step 3.1. Bring the tube and beads to 37 ˚C.
5. Plug the bead bath into an electrical outlet next to the flow cytometer in order to maintain the temperature in preparation for sample analysis.
6. 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.

4. Acquisition of calcium influx using flow cytometric analysis

1. Log into the flow cytometer software on a flow analyzer.
2. 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.
3. Click on the Acquisition tab. Open/create a new experiment and add the desired fluorescent tags to the experiment.
4. Create Reference Group and a new sample group/s for the experiment. Label both the samples and the reference groups as necessary.
5. Under the Acquisitions tab, set the events to record to the maximum: 10,000,000.
6. Set the stopping volume to the maximum volume: 3,000 µL.
7. Set the stopping time to the maximum time: 36,000 s.
8. Acquire single stained controls for defined conjugated-antibodies included in the multicolor panel.
9. Acquire single stained controls for Indo-1 functional dye.
   1. Add 50 µM 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.
   2. Add calcium free Indo-1 negative control to the flow cytometer and click on Record. EGTA was added in step 2.6.
   3. 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.
10. 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.
11. Warm single stained control samples (from steps 2.6-2.9 in section Indo-1 ratiometric dye and fluorescent antibody labeling) to 37 ˚C for sequential analysis. Set up the flow cytometry software.
12. Run DI water on the cytometer for 2-3 min to ensure the fluidic stability of the flow cytometer.
13. 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.
14. 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.
15. 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.
16. 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.
17. 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.
18. Add the next tube to the bead bath to warm to 37 ˚C.
19. 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.
20. In the Acquisition Control, click on Stop and remove the tube from Sample Injection Port (SIP).
21. Add 30 µg of unconjugated anti-CD3 directly into the sample tube to initiate calcium influx. The final concentration per tube is 60 µg/mL.
    NOTE: There is no washing step after the addition of anti-CD3.
22. Place the tube back on machine SIP as quickly as possible and press Record in the software.
23. Record data for 7 min, watching the time elapsed under the acquisition controls in the software, to observe the time-course of calcium influx within the sample populations.
24. After 7 min, press Stop in the software and add 1 µg/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.
25. Continue to the next sample and repeat the workflow until all the samples have been collected.
26. Save the experiment and click on the export zip file. Unmixed files (.fsc) are uploaded to external flow cytometry analysis software.

5. Calcium curve analysis

1. 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.
2. 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.
3. Select MFI (mean fluorescent intensity) within the Kinetics tab.
4. 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.
5. Create multiple ranges for statistics along the calcium flux time course for biological comparisons within the samples.
6. 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.
7. 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.

Wyniki

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 °C). The ...

Dyskusje

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 cytometry^7,8. 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...

Materiały

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