loading of primary mouse cd4+ t cells and coating of protein g magnetic beads with antibodies for local ca2+ imaging

<meta charset="utf8"></head><body>用于检测 T 细胞中 Ca2+ 微结构域的高分辨率成像技术

The presented fluorescence microscopy technique enables the visualization of local, temporal initial calcium (Ca2+) signals in primary mouse T cells, termed Ca2+ microdomains. Ca2+ microdomains represent highly dynamic and short-lived Ca2+ signaling events, posing challenges for effective live cell imaging and analysis1.
T cells are challenging for live cell imaging due to the relative differences in central and peripheral fluorescence intensity, which can be attributed to their spherical shape and their small diameter of ~6-8 &#181;m. Upon stimulation and immune synapse formation, T cells undergo morphological changes, further complicating the imaging of T cells1. Therefore, employing a ratiometric analysis becomes imperative, achieved by either recording two images representing different properties of a Ca2+ dye or utilizing a combination of two Ca2+ dyes. The demanding characteristics of Ca2+ microdomains include their rapid, temporally, and spatially limited nature. To capture this, the Ca2+ dyes used must possess both a high basal brightness and a high signal-to-noise ratio (SNR) to obtain the highest temporal and spatial resolution possible. Optimal results have been achieved using a combination of the dual-wavelength dye Fura Red and the single-wavelength dye Fluo-4. Co-loading cells with Fluo-4 and Fura Red mitigate the challenges posed by strong photobleaching of dual-emission wavelength dyes and the temporal delay associated with dual-excitation dyes, ensuring suitability for fast image acquisition. This approach further facilitates the visualization of changes in shape and subtle movements. Special demands are also placed on the imaging system in terms of spatial resolution to enable the visualization of Ca2+ signals originating from the opening of small clusters of channels or even single channels1.
Ca2+ signaling plays a pivotal role in activating immune functions within T cells, including synapse formation and cytokine production and release2,3. The specific fate of the cell is regulated through the differently pronounced and locally distributed Ca2+ signals, the Ca2+ microdomains3. Notably, those local Ca2+ signals precede a widespread rise in intracellular Ca2+ levels in T cells&#160;and the formation of Ca2+ microdomains depends on both Ca2+ entry and release1,4,5. Upon T cell receptor (TCR)/CD3 stimulation, the formation of Ca2+ releasing second messengers, like nicotinic acid adenine dinucleotide phosphate (NAADP), D-myo-inositol 1,4,5-trisphosphate (IP3) and cyclic ADP ribose (cADPR) is triggered, leading to rising intracellular Ca2+ levels up to 1 &#181;M6,7. Early Ca2+ signaling events are linked to the release of Ca2+ from intracellular Ca2+ stores such as the endoplasmic reticulum (ER), with channels such as the ryanodine receptor 1 (RyR1) and the IP3 receptor (IP3R) being predominantly responsible for this signaling. This subsequently triggers extracellular Ca2+ influx and results in a global Ca2+ signal via store-operated Ca2+ entry (SOCE)8. In addition, there are other channels involved in Ca2+ signaling during T cell activation9, e.g., P2X4 and P2X7 channels ensure adenosine triphosphate (ATP)-dependent cation influx, contributing to the rise in intracellular Ca2+. Remarkably, initial adhesion-dependent Ca2+ microdomains (ADCMs) are already formed&#160;before TCR stimulation but with lower Ca2+ amplitudes and frequencies. These initial TCR-independent Ca2+ signals most likely serve the migration of the T cell to the site of inflammation and prime the T cells for restimulation at the site of infection10,11.
By developing the described method for local Ca2+ imaging, we have gained an additional tool for exploring the origin and significance of early Ca2+ signals in T-cell activation. This method enables the user to detect smaller, short-lived, and more rapidly occurring Ca2+ signals than&#160;previously possible. Moreover, Deconvolution, Analysis, Registration, Tracking, and Shape normalization (DARTS), the Python-based analysis pipeline, enables sharing the analysis tools with a broader audience12.

<meta charset="utf8"></head><body>作者聚焦：揭示治疗干预的适应性免疫反应的初始激活

原代 T 细胞中初始 Ca2+ 微结构域的成像

原代小鼠 CD4+ T 细胞的上样和蛋白 G 磁珠的抗体包被，用于局部 Ca2+ 成像

loading-primary-mouse-cd4-t-cells-coating-protein-g-magnetic-beads

Research

JoVE Journal

Immunology and Infection

Loading of Primary Mouse CD4+ T Cells and Coating of Protein G Magnetic Beads with Antibodies for Local Ca2+ Imaging

179 次观看. 首先，取从小鼠淋巴结和脾脏中分离的 CD4 阳性 T 细胞。以 300 g 离心 2 至 500 万个细胞 5 分钟。弃去上清液，将沉淀重悬于 480 微升含有 10 微摩尔 Fluo-4 AM 和 20 微摩尔 Fura Red AM 的 T 细胞培养基中。用铝箔盖住 Falcon 管，并在室温下避光孵育细胞 20 分钟。然后向细胞中加入 2 mL T 细胞培养基，并继续再孵育 30 分钟。轻轻混合 Protein G 磁珠悬浮液，?...

视频: 原代小鼠 CD4+ T 细胞的上样和蛋白 G 磁珠的抗体包被，用于局部 Ca2+ 成像

Imaging Initial Ca2+ Microdomains in Primary T Cells

Local, sub-second Ca2+ signals, termed Ca2+ microdomains, are highly dynamic and short-lived Ca2+ signals, which result in a global [Ca2+]i elevation and might already determine the fate of a T cell. Upon T cell receptor activation, NAADP is formed rapidly, binding to NAADP binding proteins (HN1L/JPT2, LSM12) and their respective receptors (RyR1, TPC2) sitting on intracellular Ca2+ stores, like the ER and lysosomes, and leading to subsequent release and elevation of [Ca2+]i. To capture these fast and dynamically occurring Ca2+ signals, we developed a high-resolution imaging technique using a combination of two Ca2+ indicators, Fluo-4 AM and FuraRed AM. For postprocessing, an open-source, semi-automated Ca2+ microdomain detection approach was developed based on the programming language Python. Using this workflow, we are able to reliably detect Ca2+ microdomains on a subcellular level in primary murine and human T cells in high temporal and spatial resolution fluorescence videos. This method can also be applied to other cell types, like NK cells and murine neuronal cell lines.

Here, we provide a comprehensive protocol to resolve initial local Ca2+ signals, known as Ca2+ microdomains, in primary murine and human T cells using fluorescence microscopy. This protocol serves as a valuable resource for researchers examining Ca2+ signaling pathways within immune cells and to further unravel their function.

Local, sub-second Ca2+ signals, termed Ca2+ microdomains, are highly dynamic and short-lived Ca2+ signals, which result in a global [Ca2+]i elevation and ...