Name: analysis of t-cell receptor-induced calcium influx in primary murine t-cells by full spectrum flow cytometry
Uploaded: 2022-12-16T13:25:00
Description: 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

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.</...

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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">
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			<td>12 well TC treated plates</td>
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			<td>229111</td>
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			<td>50 mL Polypropylene centrifuge tubes with cap</td>
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			<td>Corning</td>
			<td>352052</td>
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			<td>5mL syringe</td>
			<td>BD syringe</td>
			<td>309646</td>
			<td>plunger only is used sheith is discarded</td>
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			<td>Greiner bio1</td>
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			<td>11-0081-85</td>
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			<td>Formal Scientific</td>
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			<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>
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			<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>
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			<td>DPBS 1x</td>
			<td>Gibco</td>
			<td>14190-136</td>
			<td>DPBS&#160;</td>
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			<td>EGTA</td>
			<td>Fisher</td>
			<td>NC1280093</td>
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			<td>Hyclond</td>
			<td>SH30071.03</td>
			<td>lot AE29165301</td>
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			<td>FlowJo Software</td>
			<td>https://www.flowjo.com/</td>
			<td></td>
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			<td>Indo1-AM Ester Dye</td>
			<td>ebioscience</td>
			<td>65-085-39</td>
			<td>Calcium Loading Dye&#160;</td>
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			<td>ionomycin</td>
			<td>Millipore</td>
			<td>407951-1mg</td>
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			<td>Live/Dead Ghost 540</td>
			<td>Tonbo</td>
			<td>13-0879-T100</td>
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			<td>Light Labs</td>
			<td>A-7001</td>
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			<td>Penicillin/Streptomycin/L-Glutamine</td>
			<td>Gibco</td>
			<td>10378-016</td>
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			<td>PRN694</td>
			<td>Med Chem Express</td>
			<td>Hy-12688</td>
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			<td>Purified Anti-Mouse CD16/CD32 (FC Shield) (2.4G2)</td>
			<td>Tonbo</td>
			<td>70-0161-M001</td>
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			<td>1875093</td>
			<td>&#160;+ phenol red</td>
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			<td>RPMI phenol free</td>
			<td>Gibco</td>
			<td>11835030</td>
			<td>&#160;-phenol red</td>
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			<td>Beckman Coulter</td>
			<td>Allegra612</td>
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			<td>ebioscience</td>
			<td>45-5961-82</td>
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			<td>ebioscience</td>
			<td>15-5961-82</td>
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			<td>Includes Tripan</td>
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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 ...

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