Assessing T-cell Receptor-Induced Calcium Influx Using a Calcium Indicator Dye
Published: April 30, 2024
Abstract
Source: Perrenoud, L. et al., Analysis of T-cell Receptor-Induced Calcium Influx in Primary Murine T-cells by Full Spectrum Flow Cytometry. 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 correlates with T-cell receptor-induced calcium influx.
Protocol
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 naïve mice with carbon dioxide (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 f…
Disclosures
The authors have nothing to disclose.
Materials
| 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 |
Tags
Cite This Article
Assessing T-cell Receptor-Induced Calcium Influx Using a Calcium Indicator Dye. J. Vis. Exp. (Pending Publication), e22131, doi: (2024).