Imaging Initial Ca2+ Microdomains in Primary T Cells

作者： Fynn Gerlach*1, Franziska Möckl*1, Dejan Kovacevic1,2,3, Valerie J. Brock1, Riekje Winzer4, Lina Meyer2,3, David Lohr2,3, Lena-Marie Woelk2,3, Eva Tolosa4, René Werner2,3, Björn-Philipp Diercks1 1The Calcium Signalling Group, Department of Biochemistry and Molecular Cell Biology,University Medical Centre Hamburg-Eppendorf, 2Department of Applied Medical Informatics,University Medical Centre Hamburg-Eppendorf, 3Department of Computational Neuroscience,University Medical Centre Hamburg-Eppendorf, 4Department of Immunology,University Medical Centre Hamburg-Eppendorf

简介：

Overview

This article presents a protocol for resolving initial local Ca²⁺ signals, known as Ca²⁺ microdomains, in primary murine and human T cells using fluorescence microscopy. The method enables researchers to investigate Ca²⁺ signaling pathways within immune cells, contributing to a better understanding of their functions.

Key Study Components

Area of Science

• Neuroscience
• Cell Biology
• Immunology

Background

• Calcium is a crucial intracellular signaling molecule.
• Understanding calcium activation can lead to interventions in autoimmune diseases.
• Calcium microdomains play a significant role in T cell activation.
• The study aims to link initial calcium responses to downstream signaling events.

Purpose of Study

• To decipher the complex calcium activation in T cells.
• To identify key players in calcium signaling pathways.
• To develop a method for detecting dynamic calcium microdomains in T cells.

Methods Used

• Isolation of CD4 T cells from mouse lymph nodes and spleen.
• Use of fluorescence microscopy to visualize calcium microdomains.
• Application of chemical dyes for calcium calibration.
• Measurement of calcium signals upon stimulation with antibody-coated beads.

Main Results

• Rapid formation of calcium microdomains was observed in T cells after stimulation.
• P2x4 and P2x7 mutant cells showed decreased calcium microdomain formation.
• Calcium microdomains were found at the contact sites between beads and T cells.
• The method can be adapted for other cell types and conditions.

Conclusions

• Initial calcium microdomains are critical in shaping the adaptive immune response.
• The protocol provides a valuable tool for studying calcium signaling in immune cells.
• Future work will focus on linking calcium responses to downstream events.

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