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

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…

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