assessing t-cell receptor-induced calcium influx using a calcium indicator dye

Assessing T-cell Receptor-Induced Calcium Influx Using a Calcium Indicator Dye

To begin, prepare a 1 microgram per microliter stock solution of Indo-1 AM by adding 50 microliters of DMSO into a vial, containing 50 micrograms of Indo-1 AM. Dilute the stock solution into an appropriate volume of cRPMI at a concentration of 6 micrograms per milliliter.
Place the medium in a water bath at 37 degrees Celsius. Then, use this medium to dilute a previously prepared a cell suspension at a 1-to-1 ratio to get 5 to 6 million cells per milliliter. Next, to a 12-well tissue culture plate, add 1 milliliter of cell suspension per well, per experimental condition.
Prepare a single-stained control sample for calcium-free Indo-1 AM by adding EGTA to Indo-1 dye-loaded cells. Repair a well for biological negative control with no fluorophores or Indo-1 dye. Prepare an Indo-1 positive control by adding ionomycin at a concentration of 50 micromoles to the dye-loaded cell suspension at the flow cytometer.
Also, prepare a well for single-stain controls for additional cell populations, using antibody-conjugated fluorophores. Then add 2 micrograms of FC receptor-blocking antibody per million cells, and incubate the plate for 45 minutes, with tapping every 15 minutes to resuspend the cells.
After incubation, mix and remove the entire volume of cells from the plate and add it to 1.7-milliliter microcentrifuge tubes. Centrifuge and decant the supernatant. Once the cells are washed with 1 milliliter of pre-warmed cRPMI, pellet again, and decant the supernatant.
Resuspend the cells in 1 milliliter of cRPMI, and incubate each tube, leaving the top of the tube slightly open for gas exchange, allowing complete de-esterification of the intracellular AM esters. Next, prepare a master mix of all the user-defined fluorochrome-conjugated antibodies, and add it to the resting cells for 1 milliliter of the cell volume.
After rest, pellet the cells, and wash the pellet by resuspending in 1 milliliter of cRPMI chilled to 4 degrees Celsius. Repeat this step, and place the tubes on ice. Resuspend individual samples in 500 microliters of phenol red-free cRPMI, using a 5-milliliter polystyrene flow tube with a cap.
To maintain temperature using calcium flux analysis, prepare a bead bath by adding bath beads to a small water bath, and warm it up to 37 degrees Celsius. Carefully cut a 50-milliliter conical tube in half, and discard the top of the tube. Fill 3/4 of the remaining tube with bath beads, and allow it to reach 37 degrees Celsius in the bead bath.
Place the bead bath near a flow cytometer, along with a styrofoam rack to position the tube. Before the analysis on the flow cytometer, warm the tubes individually to 37 degrees Celsius for seven minutes. Log in to the flow cytometer software, and click on the Library tab.
Then, select fluorescent tags to add calcium-bound Indo-1 and calcium-free Indo-1. Then, select UV laser under fluorescent tag groups, and click on Plus Add to add a fluorescent tag name. Choose the laser excitation and emission wavelength, and then click Save.
Continue to the experimental setup. click on the Acquisition tab. Open or create a new experiment, and add the desired fluorescent tags to the experiment. Create a reference group and a new sample group for the experiment, and label both the groups.
Under the Acquisitions tab, set the events to record to the maximum at 10 million, stopping volume to the maximum volume of 3,000 microliters, and stopping time to the maximum of 36,000 seconds. Acquire single-stained controls for user-defined conjugated antibodies, included in the multicolor panel, and for Indo-1 functional dye.
Add 50 micromoles of ionomycin to the calcium-bound Indo-1 positive control and vortex. Add the flow tube to the sample injection board, and click Record. Then, add calcium-free Indo-1 negative control to the flow cytometer, and click Record.
Unmix the single-stained controls by clicking on the Unmix button. Once unmixing is complete, create sequential plots using the Unmixed Worksheet, such as SSCA versus FSCA for removing debris from the sample, and SSCA versus SSCH to remove doublets.
Then create gates by clicking 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. Next, warm the previously prepared single-stained control samples to 37 degrees Celsius for sequential analysis.
Set up the flow cytometry software, and run deionized water for 2 to 3 minutes to ensure the fluidic stability of the flow cytometer. Set up sequential plots on the unmixed worksheet by creating gates using the polygon, rectangle, or ellipse gate buttons to Include the population of interest.
Double-click inside the gate, and change the y- and x-axis parameters to SSCA versus SSCH. Complete defining the axis parameters by left-clicking on the axis. Create a plot with SSCA versus viability dye signal. Gate the live cells, which are negative or amine dye staining.
Live cells negative for live dead staining will cluster at the zero mark on both the y- and x-axis. Double-click on the inside plot to create a new plot, containing only single-cell live lymphocytes. Change the y-axis to one versus time on the x-axis for visualization of the calcium influx.
Next, place the warmed biological sample into the SIT of the machine. Run the samples at 2,500 to 3,000 events per second on a medium flow rate to visualize calcium influx in a limited number of cell populations. Add the next tube to the bead bath.
In Acquisition control, press Record to record the initial data for 30 seconds to obtain a basal level of calcium signaling in the sample. Then click on Stop, and remove the tube from sample injection port. Add 30 micrograms of unconjugated anti-CD3 directly into the sample tube to initiate calcium influx.
Quickly place the tube back on the sample injection port, and press Record in the software. Record data for seven minutes, watching the time elapsed under the acquisition controls in the software. Then press Stop in the software.

assessing-t-cell-receptor-induced-calcium-influx-using-calcium

Research

JoVE Encyclopedia of Experiments

Immunology

Antibody-Based Technologies

Antibody-Based Imaging and Detection Methods

EoE Sub Articles

eoe sub articles

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Preparation of immune cells from the mouse spleen
NOTE: Euthanize na&#239;ve mice with carbon dioxide (CO2)&#160;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 a 50 mL conical tube with ~5 mL of complete Roswell Park Memorial Institute media (cRPMI) on ice.&#160; 
	NOTE: Complete RPMI is comprised of 10% fetal bovine serum (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&#160;x g&#160;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 ammonium&#8211;chloride&#8211;potassium (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&#160;x g&#160;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&#160;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&#160;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 dimethyl sulfoxide (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.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Do not store Indo-1 in an aqueous solution.&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
	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.&#160;&#160;&#160;&#160; 
	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 ultraviolet (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) ethylene glycol tetraacetic acid (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&#160;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&#160;x g&#160;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.&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
	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.&#160;&#160;&#160; 
		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&#160;x g&#160;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.&#160;&#160;&#160; 
	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 Dulbecco&#39;s phosphate-buffered saline (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 kills the cells at 55 &#730;C on a warming block for single stained live/dead control.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
		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 the 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.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
	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>

<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>
		<tr>
			<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>
		</tr>
		<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>

Source: Perrenoud, L. et al., <a href="https://app.jove.com/v/64526">Analysis of T-cell Receptor-Induced Calcium Influx in Primary Murine T-cells by Full Spectrum Flow Cytometry.</a>&#160;J. Vis. Exp. (2022)
This video analyzes T-cell receptor-induced calcium influx in T-cells. Anti-CD3 antibodies activate the T-cell signaling pathways, leading to an increase in intracellular calcium ions that bind with Indo-1 dye. In flow cytometry, a spectrum shift from the calcium-free state to the calcium-bound state&#160; correlates with T-cell receptor-induced calcium influx.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Begin with a mouse T cell suspension and introduce a calcium indicator dye &#8212; Indo-1-acetoxymethyl ester.
The dye enters the cells, where intracellular esterases cleave the acetoxymethyl group to form membrane-impermeable Indo-1, which exhibits distinct fluorescence in calcium-free and calcium-bound forms.&#160;
Add Fc receptor antibodies to block nonspecific interaction sites.
Introduce fluorophore-labeled antibodies to stain T cell subsets.
Remove unbound antibodies and stain with a viability dye that differentially stains live and dead cells.&#160;
In flow cytometry, initially detect dim fluorescence of live cells and identify fluorophore-tagged T cells, followed by measuring Indo-1 fluorescence within these cells.
Treat with anti-CD3 antibodies, which interact with the T cell receptor CD3 complex and activate T cell signaling pathways, inducing calcium influx into the cytosol.
Indo-1 dye interacts with the released calcium ions, shifting the dye&#39;s fluorescence from blue to violet.
The shift in Indo-1 fluorescence from calcium-free to calcium-bound states confirms T cell receptor-induced calcium influx.

23 조회수. 칼슘 지시약 염료를 사용한 T 세포 수용체 유도 칼슘 유입 평가

비디오: 칼슘 지시약 염료를 사용한 T 세포 수용체 유도 칼슘 유입 평가 - 실험

Spatial and Temporal Control of T Cell Activation Using a Photoactivatable Agonist

T lymphocytes engage in rapid, polarized signaling, occurring within minutes following TCR activation. This induces formation of the immunological synapse, a stereotyped cell-cell junction that regulates T cell activation and directionally targets effector responses. To study these processes effectively, an imaging approach that is tailored to capturing fast, polarized responses is necessary. This protocol describes such a system, which is based on a photoactivatable peptide-major histocompatibility complex (pMHC) that is non-stimulatory until it is exposed to ultraviolet light. Targeted decaging of this reagent during videomicroscopy experiments enables precise spatiotemporal control of TCR activation and high-resolution monitoring of subsequent cellular responses by total internal reflection (TIRF) imaging. This approach is also compatible with genetic and pharmacological perturbation strategies. This allows for the assembly of well-defined molecular pathways that link TCR signaling to the formation of the polarized cytoskeletal structures that underlie the immunological synapse.

This protocol describes an imaging-based method to activate T lymphocytes using photoactivatable peptide-MHC, enabling precise spatiotemporal control of T cell activation.

Photoactivatable 길 항 제를 사용 하 여 T 세포 활성화의 공간 및 시간 제어

T lymphocytes engage in rapid, polarized signaling, occurring within minutes following TCR activation. This induces formation of the immunological ...

연구

JoVE Journal

면역학 및 감염병학

An In Vivo Mouse Model to Measure Na&#239;ve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells

Quantification of na&#239;ve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played by T cells in an immune response. This protocol describes the in vitro differentiation of bone marrow (BM) progenitors to obtain granulocyte macrophage colony-stimulating factor (GM-CSF) derived-dendritic cells (DCs). The protocol also describes the adoptive transfer of ovalbumin peptide (OVAp)-loaded GM-CSF-derived DCs and na&#239;ve CD4 T cells from OTII transgenic mice in order to analyze the in vivo activation, proliferation, and Th1 differentiation of the transferred CD4 T cells. This protocol circumvents the limitation of purely in vivo methods imposed by the inability to specifically manipulate or select the studied cell population. Moreover, this protocol allows studies in an in vivo environment, thus avoiding alterations to functional factors that may occur in vitro and including the influence of cell types and other factors only found in intact organs. The protocol is a useful tool for generating changes in DCs and T cells that modify adaptive immune responses, potentially providing important results to understand the origin or development of numerous immune associated diseases.

Here, we present a protocol for the in vivo determination of na&#239;ve CD4 T cell (T cell) activation, proliferation, and Th1 differentiation induced by GM-CSF bone marrow (BM)-derived dendritic cells (DCs). In addition, this protocol describes BM and T-cell isolation, DC generation, and DC and T-cell adoptive transfer.

Vivo에서 마우스 모델을 측정 순진한 CD4 T 세포 활성화, 확산 및 골 수 유래 수지상 세포에 의해 유도 된 Th1 차별화

Quantification of na&#239;ve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played ...

Antigenic Liposomes for Generation of Disease-specific Antibodies

Antibody responses provide critical protective immunity to a wide array of pathogens. There remains a high interest in generating robust antibodies for vaccination as well as understand how pathogenic antibody responses develop in allergies and autoimmune disease. Generating robust antigen-specific antibody responses is not always trivial. In mouse models, it often requires multiple rounds of immunizations with adjuvant that leads to a great deal of variability in the levels of induced antibodies. One example is in mouse models of peanut allergies where more robust and reproducible models that minimize mouse numbers and the use of adjuvant would be beneficial. Presented here is a highly reproducible mouse model of peanut allergy anaphylaxis. This new model relies on two key factors: (1) antigen-specific splenocytes are adoptively transferred from a peanut-sensitized mouse into a na&#239;ve recipient mouse, normalizing the number of antigen-specific memory B- and T-cells across a large number of mice; and (2) recipient mice are subsequently boosted with a strong multivalent immunogen in the form of liposomal nanoparticles displaying the major peanut allergen (Ara h 2). The major advantage of this model is its reproducibility, which ultimately lowers the number of animals used in each study, while minimizing the number of animals receiving multiple injections of adjuvant. The modular assembly of these immunogenic liposomes provides relatively facile adaptability to other allergic or autoimmune models that involve pathogenic antibodies.

Described is the preparation of antigenic liposomal nanoparticles and their use in stimulating B-cell activation in vitro and in vivo. Consistent and robust antibody responses led to the development of a new peanut allergy model. The protocol for generating antigenic liposomes can be extended to different antigens and immunization models.

질병-특정 항 체의 생성에 대 한 항 원 리

Antibody responses provide critical protective immunity to a wide array of pathogens. There remains a high interest in generating robust antibodies for ...

Assessing T-cell Receptor-Induced Calcium Influx Using a Calcium Indicator Dye

내레이션 대본

Assessing T-cell Receptor-Induced Calcium Influx Using a Calcium Indicator Dye