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…
Declarações
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