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>T hücrelerinde Ca2+ mikro alanları tespit etmek için yüksek çözünürlüklü görüntüleme tekniği

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>Yazar Spot Işığı: Terapötik Müdahale için Adaptif Bağışıklık Tepkisinin İlk Aktivasyonunun Çözülmesi

Primer T Hücrelerinde İlk Ca2+ Mikro Alanlarının Görüntülenmesi

Our fundamental research, we want to understand the initial activation of the adaptive immune response. Calcium is one of the most widely used intracellular signal messengers. If we decipher the complex calcium activation, we may be able to intervene with a signaling machinery to treat pathological conditions such as autoimmune disease.Using this method, we were able to identify the key players in the NADP signal in cascade and T cells, such as the NADP producing enzyme and NADP binding protein. Moreover, we were able to show that adhesion dependent calcium micro domains contribute to initial calcium signals independent of T-cell receptor stimulation. Our method is the first capable of detecting small, fast and highly dynamic calcium micro domains in primary T-cells, and can be easily adapted to other cell types, or stimulations and inhibitions.Additionally, the combination of two chemical dyes allows the calibration of these calcium micro remains. We believe that the initial calcium micro remains shape the adaptive immune response by controlling the fate of individual T cells. Therefore, want to link the initial calcium response to downstream cycling events and adapt our imaging to life cell super resolution to resolve initial calcium nano domains.

Primer Fare CD4+ T Hücrelerinin Yüklenmesi ve Protein G Manyetik Boncukların Lokal Ca2+ Görüntüleme için Antikorlarla Kaplanması

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

127 Görüntüleme. Başlamak için, fare lenf düğümlerinden ve dalağından izole edilen CD4 pozitif T hücrelerini alın. İki ila beş milyon hücreyi 300 g'da beş dakika boyunca santrifüjleyin. Süpernatanı atın ve peleti 10 mikromolar Fluo-4 ve 20 mikromolar Fura Red içeren 480 mikrolitre T hücresi ortamında yeniden süspanse edin. Falcon tüpünü alüminyum folyo ile örtün ve hücreleri karanlıkta oda sıcaklığında 20 dakika inkübe edin.Daha sonra ...

Video: Primer Fare CD4+ T Hücrelerinin Yüklenmesi ve Protein G Manyetik Boncukların Lokal Ca2+ Görüntüleme için Antikorlarla Kaplanması

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 ...

İmmünoloji ve Enfeksiyon

To begin, take CD4-positive T cells isolated from mouse lymph nodes and spleen. Centrifuge two to five million cells for five minutes at 300 g. Discard the supernatant and resuspend the pellet in 480 microliters of T cell medium containing 10 micromolar Fluo-4 AM and 20 micromolar Fura Red AM.Cover the Falcon tube with aluminum foil and incubate the cells for 20 minutes at room temperature in the dark.Then add two milliliters of T cell medium to the cells and continue incubating for an additional 30 minutes. Gently mix Protein G magnetic bead suspension and transfer 12.5 microliters to a new tube. Place the tube on a magnetic stand to allow the beads to migrate to the magnet.Gently pipette out the storage buffer from the opposite side of the magnet to remove it. To remove any remaining storage buffer, add 500 microliters of PBST and vortex for 10 seconds. Place the tube on the magnetic stand again and remove the buffer.To coat the beads with antibodies, resuspend them in 7.5 microliters of PBST and add five microliters each of anti-CD3 and anti-CD28. Incubate for 30 to 60 minutes while continuous mixing at room temperature. Wash the coated beads three times with 500 microliters of PBST, followed by a wash with 500 microliters of calcium buffer using the magnetic stand, then resuspend the beads in 200 to 400 microliters of calcium buffer.

Detecting Ca2+ Microdomains on a Subcellular Level in Primary Murine T Cells Using Fluorescence Microscopy

To begin, place 10 microliters of the loaded CD4+cells on the prepared slide and let them attach for three to five minutes. Gently add 80 microliters of calcium measuring buffer to the slide. Select the 100X oil immersion lens.Apply a small drop of immersion oil and place the slide on the microscope stage. Then adjust the focus in brightfield mode. Select a field-of-view with up to 10 cells that are not in contact with each other and acquire an image.Next, add 10 microliters of antibody-coated beads or stimulant after one minute, and measure for a total of three minutes using 40 frames per second or the maximum frame rate possible. Use the slider to pinpoint the time of contact between a bead and a cell of interest. In the Options menu on the right hand side, select bead contact:x, y, t.Select the spot on the left side of the image where the cell and bead make contact. Select Choose cell by clicking a point inside. Click on the cell stimulated by this bead, preferably in the center.Click on ADD bead contact. Once all bead contacts are defined, click on the Continue button and proceed with additional files Wild-type cells displayed rapid calcium microdomain formation within the first second after stimulation, which expanded throughout the cell over the subsequent 15 seconds. Conversely, P2x4 and P2x7 mutant cells showed decreased calcium microdomain formation with P2x4 mutant cells also presenting a lower basal level prior to stimulation.Like murine T cells, calcium microdomains were observed at the bead contact site in human CD4+T cells.