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 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.
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.
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.
2. Indo-1 ratiometric dye and fluorescent antibody labeling
3. Bead bath tube: maintaining temperature during calcium flux analysis
4. Acquisition of calcium influx using flow cytometric analysis
5. Calcium curve analysis
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 samples are then analyzed using full spectrum flow cytometry for Indo-1 fluorescence after gating on the individual cell types desired, without the need for sorting the samples prior to analysis. In order to use Avalanche Photo Diode (APD) detectors within the full spectrum flow cytometer, it is necessary to convert the peak emission wavelength from a bandpass filter wavelength measured in nm to its respective channel on the APD. The table shown in Figure 2 provides a guideline, but the optimum detectors for the two fluorescence signatures of Indo-1 (calcium-bound versus calcium-free) must be determined empirically. This is accomplished by preparing single stained Indo-1 samples using Ionomycin to generate the Indo-1 (calcium-bound) signal, and EGTA, a calcium chelating agent, to generate the Indo-1 (calcium-free) signal. After analysis using full spectrum flow cytometry, the channel displaying the peak fluorescence intensity for each Indo-1 signature can be visualized (Figure 3A). By normalizing the MFI of the positive populations to that of the negative populations, the shift in Indo-1 fluorescence from calcium-free to calcium-bound can be determined (Figure 3B). This display is generated in a workbook (.xls) using reference controls for both bound and free Indo-1, drawing a negative and positive interval gate with clear positive and negative gates for MFI normalization as shown in (Figure 3A). Sample MFI statistics can also be obtained by right clicking on the stats table. In the software, determine the MFI ratio by dividing the positive population with the negative population and overlay the two spectra on a single display (Figure 3B, right). The yellow dotted arrow shows the normalized spectral differences between the spectral signatures of both bound and free Indo-1.
To evaluate calcium responses following T-cell receptor stimulation, the Indo-1 fluorescence is assessed after gating on the cell populations of interest, as shown in Figure 4. First, a plot of time versus side scatter (SSC-A) is examined to assess fluidic stability; a stable consistent signal across the time axis indicates stability (Figure 4A). Next, the forward versus side scatter (FSC-A vs. SSC-A) is visualized, and the lymphocyte population is gated as shown in (Figure 4B). After the lymphocyte gate, a single cell discrimination gate is applied by generating a plot of side scatter height versus area (SSC-H vs. SSC-A), and the singlets, which fall on the diagonal, are gated as shown in Figure 4C. The singlets are then assessed for viability, by examining staining with the viability dye and gating on the negative population (Figure 4D). Finally, to analyze T-cells, a B-cell dump/internal negative control gate is applied by gating on the CD19-negative population (Figure 4E). The CD19-negative population is then assessed for T-cell subsets using antibodies to CD4 and CD8 (Figure 4F); the example shown examines CD4+ T-cells for calcium responses by visualizing Indo-1 (calcium-bound) fluorescence versus time (Figure 4G). After gating on a time segment, the Indo-1 (calcium-free) versus Indo-1 (calcium-bound) plot can also be generated (Figure 4H). This analysis will later be used to derive the Indo-1 ratio (see methods calcium influx curve analysis in flow cytometry analysis software).
At this point in the analysis, it may be useful to analyze moments in time within the two-dimensional plot of Indo-1 (calcium-bound) versus time in individual cell populations of interest. The optimal timepoints include the baseline calcium bound to Indo-1 prior to TCR stimulation, the peak response, a timepoint several minutes after the peak response as intracellular calcium levels decline, and finally the calcium response elicited by adding ionomycin (Figure 5A). In the baseline sample prior to anti-CD3 antibody addition, a weak Indo-1 (calcium-bound) signal is detected. During the peak response, the cells show a strong Indo- 1(calcium-bound) signal and a reduced fluorescence of Indo-1 (calcium-free). At 5 min post-peak, the Indo-1 fluorescence nearly returned to baseline. After ionomycin is added to the sample, a robust signal of Indo-1 (calcium-bound) can be visualized, ensuring adequate dye-loading of the cells. Rare cell populations (<1 %) can be difficult for derivative analysis; to overcome this limitation, analysis of the rare populations can be performed by plotting Indo-1 (calcium-bound) versus Indo-1 (calcium free) after gating on each population of interest, such as CD4+ cells, TCRb+ cells, TCRyg+ cells, CD25+ cells, iNKT cells, and CD19+ cells (Figure 5B,C). Note the readily detectable calcium response in TCRγδ+ T-cells and iNKT cells (CD1d/α-galcer+). For comparison of surface proteins within a mixed murine T-cell population, two-dimensional plots were created showing subsets of interest. Below, the analysis of the calcium response over the entire time course for each subset is displayed (Figure 5C).
This assay can also be used to establish biological differences in T-cells from wild-type versus gene-targeted mice or in T-cells untreated versus treated with pharmacological inhibitors. In the example shown in Figure 6, T-cells were treated with PRN694, a small molecule inhibitor of the T-cell tyrosine kinases, ITK and RLK9,10. Inhibition of ITK/RLK dampens T-cell receptor signaling, leading to a reduced calcium response following T-cell receptor stimulation11 (Figure 6A). To quantify the effects of ITK/RLK inhibition, the curves displaying the ratio of Indo-1 (calcium-bound) to Indo-1 (calcium-free) fluorescence can then be analyzed for the area under the curve (AUC) or slope of the curve for each condition (Figure 6B,C). This quantification is performed by sectioning the time course of the calcium response into discrete segments (Figure 6A), and assessing the area under the curve or the slope of the curve for each segment, as shown (Figure 6B,C). Overall, this protocol demonstrates that full spectrum flow cytometry can be used to measure calcium influx along with multiple cell surface markers in immune cell populations under investigation. These results show that cell subsets represented at greater than 1% of the total population can be assessed for calcium influx over time. In contrast, less abundant cell subsets require alternative analysis using moments of time.
Statistical analysis of calcium response data is specific to the experimental question and the biological assays that are being performed. In the data presented in the manuscript, experiments were performed with replicates to ensure experimental accuracy and the robustness of the methodology; however, multiple experiments on different biological samples over different days were not performed. Therefore, it would not be appropriate to perform statistical analyses on the data presented.
Figure 1: Schematic of calcium assay workflow. The use of the Indo-1 AM ester calcium indicator dye provides a tool to visualize the influx of calcium in immune cells and their sub-populations. This schematic outlines the workflow for analysis of calcium responses in murine splenic T-cells. Please click here to view a larger version of this figure.
Figure 2: Conversion of PMT wavelengths to APD channels. The full spectrum flow cytometer has 16 detectors to detect fluorescent emissions following excitation by the UV laser. The specifications of each detector are indicated in the table. Highlighted are the two detectors used for the assessment of Indo-1 fluorescence, as were determined empirically using single stained controls. Conversions to APD wavelength and user manual for spectral flow cytometer are available11. Please click here to view a larger version of this figure.
Figure 3: Visualization of the Indo-1 spectral signature. (A) Top: Spectral signature of Indo-1 fluorescence in CD8+ T-cells following ionomycin introduction to elicit a robust influx of calcium. The peak of Indo-1 (calcium-bound) can be visualized in UV1. Bottom: Spectral signature of Indo-1 (calcium-free) showing the peak fluorescence in UV7. This control sample was generated by treating Indo-1-loaded cells with EGTA12 to chelate any extracellular free calcium. (B) Combined five laser spectrum overlay of the Indo-1 signature, showing raw spectral MFI on the left and the normalized spectra of Indo-1 bound versus free Indo-1 on the right. Visualization of the shift from Indo-1 (calcium-free) in orange to Indo-1 (calcium-bound) in blue can be readily detected. Please click here to view a larger version of this figure.
Figure 4: Gating strategy for visualization of calcium responses. The workflow for sequential gating of the samples is outlined. (A) Fluidic stability is examined using a time versus SSC-A gate. (B) FSC-A versus SSC-A is used to gate on lymphocytes, thereby eliminating cell debris and residual red blood cells in the sample. (C) Single cells are gated on using a SSC-H versus SSC-A plot. (D). The singlet gate is then applied to a plot of live-dead Ghost540 versus SSC-A; gating on Ghost540-negative cells eliminate non-viable cells from subsequent analysis. (E) Live cells are analyzed for CD19 versus SSC-A, and CD19-negative cells (non-B cells) are gated on. (F) The CD19-negative cells are examined for CD4 versus CD8 staining. (G) CD4+ cells are then gated on to visualize time versus Indo-1 (calcium-bound). (H) A moment in time is selected to represent the initiation of the calcium influx response following anti-CD3 antibody addition by visualizing Indo-1 (calcium-free) versus Indo-1 (calcium-bound) fluorescence. Please click here to view a larger version of this figure.
Figure 5: Visualization of Indo-1 fluorescence in gated populations of murine thymocytes at discrete timepoints in the calcium response. Total thymocytes were stimulated with anti-CD3 antibody followed by ionomycin after 6 min of the sample collection. At four discrete timepoints, as indicated in the rectangular boxes outlined on the two-dimensional plots of time versus Indo-1 (calcium-bound), different subpopulations of thymocytes were examined for calcium responses. (A) Two-dimensional plots showing gating for the baseline Indo-1 signal, the peak response, 5 min post-peak response, and the response to ionomycin. Below each plot CD4 versus CD8 staining (left) and the plot of Indo-1 (calcium-free) versus Indo-1 (calcium-bound) on gated CD4 single positive (SP) thymocytes is shown. (B) Plots of Indo-1 (calcium-free) versus Indo-1 (calcium-bound) on gated subsets of thymocytes are shown at each of the four time-points, using the gating strategy shown in (A). (C) Two-dimensional plots were created showing staining of total thymocytes with the indicated antibodies. Specific subsets were gated (top row) on and examined for calcium responses versus time (bottom). Light blue (SP) CD8+ population, red (SP) CD4 population, which exhibits the greatest influx of calcium, orange CD4+CD8+ double-positive thymocytes, dark green iNKT cells, light green CD25+, purple TCRγδ+ T-cells, and black CD19+ B cells. Please click here to view a larger version of this figure.
Figure 6: Inhibition of the calcium response in CD8+ T-cells treated with a small molecule inhibitor of ITK/RLK. During the Indo-1 dye-loading, splenocytes were treated with two doses of PRN694, 100 nM (dark gray) and 200 nM (light gray). (A) Indo-1 ratio (calcium-bound/calcium-free) is shown versus time for untreated cells (red) and cells treated with PRN694. Curves show calcium response of gated CD8+ T-cells. The black line represents the negative control of splenocytes loaded with Indo-1 and surface stained, but not stimulated with anti-CD3 antibody. (B,C) The data can be analyzed by dividing the time axis shown in (A) into eight segments, visualized with black dotted lines, and calculating the area under the curve (AUC) (B), or the slope of each curve (slope) (C) at each time interval of the response. Please click here to view a larger version of this figure.
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 highly overlapping dyes, thereby increasing the number of markers that can be used in a panel. This is due to the fact that the full spectrum flow cytometer collects all of the laser light emitted across an array of detectors for each sample analyzed, rather than having one detector dedicated to one fluorochrome. Using the entire spectrum of each fluor allows for higher sensitivity of the assay and provides a means to identify spectrally unique fluorochromes that would have overlapping signals if collected on a band-pass flow cytometer. This also eliminates the need for pre-isolation of a cell subset under investigation.
In this study, a panel of cell surface markers were assessed with fluorochrome-conjugated antibodies. This panel included αCD4-APC-Cy7, αCD8-FITC, αCD19-PE, αTCRβ-PerCP-Cy5.5, α-TCRδ-PE-Cy5, αCD25-PE-Cy7, and CD1d-αgal-cer tetramer-APC; in addition, the panel included the live-dead Ghost540 dye. The optimal channel to detect each of these fluorochromes is available in the full spectrum flow cytometer users manual13. In contrast, the detection of Indo-1 (calcium-free) and Indo-1 (calcium bound) was empirically determined, although an approximate peak channel for detection of each fluorescent signature was estimated based on the known peak emission wavelengths for the two forms of Indo-1. It is also possible to convert the peak emission wavelengths from bandpass filter wavelengths used on conventional flow cytometers and measured in nm to their respective channels on the Avalanche Photo Diode (APD) detectors used by the full spectrum flow cytometer; this can also provide an estimate of optimal channels to detect Indo-1 (calcium-free) and Indo-1 (calcium bound), respectively. Since single stained controls are included for each fluorescent tag, the unmixing process deconvolutes the fluorescent signatures, allowing visualization of each fluorochrome's intensity on cells in each sample. As described in this method, data analysis can be performed by gating on cell subsets of interest and visualizing the ratio of detect Indo-1 (calcium-free) and Indo-1 (calcium bound) versus time.
There are limitations of this assay. For instance, concentrations of Indo-1 below 3 µM did not allow for successful visualization of the calcium influx response in T-cells. As this assay also accommodates multiplexing measurements of the calcium response with analysis of multiple cell surface markers on heterogeneous populations of cells using full spectrum flow cytometry, it is imperative to carefully titrate all fluorescently tagged antibodies used, including those used for the single stained controls. This is important for successful implementation of the highly sensitive linear unmixing algorithm14. In addition, a complete time course of calcium influx could not be visualized for cell subsets representing less than 1% of the total population being analyzed. Instead, an assessment of the calcium response in these rare subsets required an alternative analysis strategy using moments in time15. The moments in time analysis provides snapshots of the fluorescent signals of Indo-1 (calcium-free) versus Indo-1 (calcium bound) at discrete stages of the calcium response, rather than the entire time course of the response. Finally, it is also important to recognize the limitations of using single surface markers to define subsets of T-cells. For instance, staining thymocytes with α-CD25 antibody is not sufficient to distinguish CD4+ regulatory T-cells from all other thymocyte populations. This is due to the fact that the majority of early thymocyte progenitor (CD4-CD8-) cells also express CD2516. This is likely to account for the absence of a detectable calcium influx response in gated CD4+CD25+ thymocytes.
The optimization of this protocol required careful assessment of several variables that were key to assay success. These include optimization of the specific clone of anti-CD3 antibody used, titration of the optimal concentration of this antibody, and the need for antibody crosslinking using the traditional biotin/streptavidin system. For murine T-cells, the concentration of 30 µg/sample for 6 x 106 cells in a 500 µL volume was found to be the minimum amount of antibody necessary for optimal calcium responses. Interestingly, robust calcium responses were observed with anti-CD3 antibody clone 17A2, but not with clone 145-2C11; furthermore, when using clone 17A2, adding a secondary crosslinking reagent was unnecessary17. Tests using biotinylated anti-CD3 clone 17A2 with or without streptavidin showed no enhancement of the calcium response in the presence of streptavidin crosslinking. It is possible that this antibody included aggregates, and that these aggregates accounted for the stimulation efficacy of the antibody in the absence of overt cross-linking. However, as these experiments were performed over the course of many months with different vials of this anti-CD3 antibody, the results obtained were highly reproducible; therefore, the ability of this antibody to stimulate T-cells is not likely to vary among users.
Robust calcium responses in primary T-cells require maintaining the cells at the biological temperature of 37 ˚C throughout the sample acquisition; in contrast, primary B cells will show a calcium response to anti-IgM antibody at room temperature. Based on the sensitivity of the T-cell calcium response to temperature, it is critical to ensure that each sample is treated the same; for instance, each sample must be warmed to 37 ˚C using the same method and for the same length of time. In the absence of this consistency, variations in calcium responses may be observed, but may not be indicative of biologically relevant differences between samples.
In summary, this protocol describes the details of performing calcium flux assays based on the assessment of Indo-1 fluorescence using full spectrum flow cytometry in combination with multiplex surface cell marker staining. The method allows investigators to avoid the need for isolation or sorting of specific cell populations prior to calcium dye labeling. Furthermore, this protocol outlines an additional analysis methodology used to visualize calcium responses at discrete moments in time within distinct cell subpopulations. The moments in time analysis can be successfully applied to rare (<1% of total) cell populations, which are otherwise undetectable when assessing the entirety of the calcium response curve. In future, this assay could be expanded to the use of additional fluorochrome-conjugated antibodies, taking advantage of the extensive capabilities of the full spectrum flow cytometer.
The authors have nothing to disclose.
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.
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 |