Abkürzungen: ACC - Adrenal chromaffinen Zellen, E. Soln - Enzymlösung, E. DMEM - Enriched Dulbeccos Modified Eagle Medium <ol><li> Vorbereitung <ol><li> Add Papain auf die gewünschte Menge an E. Soln (40 Einheiten Papain / 1ml E. Soln.). </li><li> Aktivieren Papain mit Carbogen für etwa 15 Minuten auf dem Eis. Oxygenat Lockes Puffer auf Eis als auch. </li></ol></li><li> Zergliederung <ol><li> Verwenden Sie 8-10 Wochen Maus. </li><li> Vor Verdauung entfernen Sauerstoff Lockes Puffer und auf Eis stellen. </li><li> Siehe Video zum Präparieren Verfahren. </li><li> Einmal ausgeschnitten, mit Sauerstoff statt Drüsen in Lockes Puffer. </li></ol></li><li> Vorbereitung von Gland <ol><li> Entfernen Sie Fett aus Drüse. </li><li> Streifen Rinde ab Drüse. </li></ol></li><li> Enzymatische Verdauung <ol><li> Sterilfilter Sauerstoff Papain. </li><li> Machen Sie vier Pools des oxygenierten Papain auf eine Petrischale. <ol><li> 3 Pools sollte etwa 100uls werden. </li><li> 1 des Pools sollte etwa 400uls werden. </li></ol></li><li> Wash medullae durch Pools <ol><li> 1. bis der 3 100 ul-Pools. </li><li> Dann durch die 400ul Pool </li><li> Pippette 300ul E. Soln und Medulla Stücke aus 400ul Pool in Mikrozentrifugenröhrchen. </li><li> Qrap Parafilm auf Mikrozentrifugenröhrchen und in 37 ° C Wasserbad für 20min. </li><li> Denken Sie daran, mit Sauerstoff zu versorgen restlichen E. Soln mit Papain fortsetzen. </li><li> Nach 20 Minuten Sterilfilter mehr E. Soln. </li><li> Ersetzen Sie alte E. Soln Baden medullae mit neu gefiltert E. Soln. </li></ol></li></ol></li><li> Verreibungen <ol><li> Absaugen und entsorgen E. Soln. </li><li> Wash medullae in 1ml E. DMEM. </li><li> Absaugen und entsorgen E. DMEM. </li><li> Add 300ul E. DMEM und verreiben 20X mit 1ml Spitze. </li><li> Cut 200 &amp;mgr; l Spitze mit einer sterilen Rasierklinge. </li><li> Man reibt 5-10X mit Ausschnitt 200 &amp;mgr; l Spitze. </li><li> Discard E. DMEM Baden medullae Stücke. Medium enthält Schutt. </li><li> Add 300uls frischen E. DMEM in Stücke medullae. </li><li> Man reibt mit 200 &amp;mgr; l Spitze 20x </li><li> Pippette E. DMEM Mikrozentrifugenröhrchen withough medullae Stücke. Medium enthält freie ACC. </li><li> Add 300uls frischen E. DMEM in Stücke medullae. </li><li> Man reibt medullae mit einem 23,5 gauge Kanüle 20x. </li><li> Kombinieren E. DMEM aus beiden Verreibungen. Medullae Stücke sollten nicht anwesend sein und sollten nun eine federähnliche Aussehen. </li></ol></li><li> Plating <ol><li> Microfuge E. DMEM aus den letzten beiden Verreibung Schritte für 2,5 Minuten bei 3,7 g s. </li><li> Pellet in 50uls-200 E. DMEM, abhängig von nachfolgenden Experimenten. </li><li> Platte 10-30uls auf einem Deckglas. </li><li> Warten Sie 15 Minuten für die Zellen zu haften. </li><li> Add 750uls von E. DMEM. </li></ol></li></ol>

The authors have nothing to disclose.

mouse adrenal chromaffin cell isolation

Nebennierenmarks chromaffinen Zellkultur-Systeme sind äußerst nützlich für die Untersuchung der Anregungs-Sekretion Kopplung in einem in vitro-Einstellung. Dieses Protokoll stellt das Verfahren verwendet werden, um den Nebennieren zu sezieren und dann isolieren medulläre Region durch Abstreifen der Nebennierenrinde. Die Verdauung der Medulla in einzelne chromaffinen Zellen wird dann demonstriert.

Watch this Scientific Journal Video about Mouse Adrenal Chromaffin Cell Isolation at JoVE.com

Mouse Adrenal Chromaffin Cell Isolation

Maus Adrenal Chromaffin Zellisolation

Adrenal medullary chromaffin cell culture systems are extremely useful for the study of excitation-secretion coupling in an in vitro setting.  This ...

Research

JoVE Journal

Biology

17.1K Aufrufe.  University of California, Irvine (UCI). Note: This video does n

Serie: Mouse Adrenal Chromaffin Cell Isolation

Methods for Cell-attached Capacitance Measurements in Mouse Adrenal Chromaffin Cell

Neuronal transmission is an integral part of cellular communication within the brain. Depolarization of the presynaptic membrane leads to vesicle fusion known as exocytosis that mediates synaptic transmission. Subsequent retrieval of synaptic vesicles is necessary to generate new neurotransmitter-filled vesicles in a process identified as endocytosis. During exocytosis, fusing vesicle membranes will result in an increase in surface area and subsequent endocytosis results in a decrease in the surface area. Here, our lab demonstrates a basic introduction to cell-attached capacitance recordings of single endocytic events in the mouse adrenal chromaffin cell. This type of electrical recording is useful for high-resolution recordings of exocytosis and endocytosis at the single vesicle level. While this technique can detect both vesicle exocytosis and endocytosis, the focus of our lab is vesicle endocytosis. Moreover, this technique allows us to analyze the kinetics of single endocytic events. Here the methods for mouse adrenal gland tissue dissection, chromaffin cell culture, basic cell-attached techniques, and subsequent examples of individual traces measuring singular endocytic event are described.

After exocytosis, fused plasma membrane is retrieved through a process known as endocytosis. This mechanism reforms new synaptic vesicles for the next round of release. Individual endocytic events are captured and analyzed through the use of the cell-attached capacitance recordings in mouse adrenal chromaffin cells.

Methoden für die Cell-Attached-Kapazitätsmessungen in Maus-chromaffinen Nebennierenzell

Neuronal transmission is an integral part of cellular communication within the brain. Depolarization of the presynaptic membrane leads to vesicle fusion ...

Forschung

Neurowissenschaften

Isolation, Fixation, and Immunofluorescence Imaging of Mouse Adrenal Glands

Immunofluorescence is a well-established technique for detection of antigens in tissues with the employment of fluorochrome-conjugated antibodies and has a broad spectrum of applications. Detection of antigens allows for characterization and identification of multiple cell types. Located above the kidneys and encapsulated by a layer of mesenchymal cells, the adrenal gland is an endocrine organ composed by two different tissues with different embryological origins, the mesonephric intermediate mesoderm-derived outer cortex and the neural crest-derived inner medulla. The adrenal cortex secretes steroids (i.e., mineralocorticoids, glucocorticoids, sex hormones), whereas the adrenal medulla produces catecholamines (i.e., adrenaline, noradrenaline). While conducting adrenal research, it is important to be able to distinguish unique cells with different functions. Here we provide a protocol developed in our laboratory that describes a series of sequential steps required for obtaining immunofluorescence staining to characterize the cell types of the adrenal gland. We focus first on the dissection of the mouse adrenal glands, the microscopic removal of periadrenal fat followed by the fixation, processing and paraffin embedding of the tissue. We then describe sectioning of the tissue blocks with a rotary microtome. Lastly, we detail a protocol for immunofluorescent staining of adrenal glands that we have developed to minimize both non-specific antibody binding and autofluorescence in order to achieve an optimal signal.

Here we present a method to isolate adrenal glands from mice, fix the tissues, section them, and perform immunofluorescence staining.

Isolierung, Fixierung und Immunfluoreszenz Imaging der Maus Nebennieren

Immunofluorescence is a well-established technique for detection of antigens in tissues with the employment of fluorochrome-conjugated antibodies and has ...

Immunologie und Infektion

Monitoring the Effect of Osmotic Stress on Secretory Vesicles and Exocytosis

Amperometry recording of cells subjected to osmotic shock show that secretory cells respond to this physical stress by reducing the exocytosis activity and the amount of neurotransmitter released from vesicles in single exocytosis events. It has been suggested that the reduction in neurotransmitters expelled is due to alterations in membrane biophysical properties when cells shrink in response to osmotic stress and with assumptions made that secretory vesicles in the cell cytoplasm are not affected by extracellular osmotic stress. Amperometry recording of exocytosis monitors what is released from cells the moment a vesicle fuses with the plasma membrane, but does not provide information on the vesicle content before the vesicle fusion is triggered. Therefore, by combining amperometry recording with other complementary analytical methods that are capable of characterizing the secretory vesicles before exocytosis at cells is triggered offers a broader overview for examining how secretory vesicles and the exocytosis process are affected by osmotic shock. We here describe how complementing amperometry recording with intracellular electrochemical cytometry and transmission electron microscopy (TEM) imaging can be used to characterize alterations in secretory vesicles size and neurotransmitter content at chromaffin cells in relation to exocytosis activity before and after exposure to osmotic stress. By linking the quantitative information gained from experiments using all three analytical methods, conclusions were previously made that secretory vesicles respond to extracellular osmotic stress by shrinking in size and reducing the vesicle quantal size to maintain a constant vesicle neurotransmitter concentration. Hence, this gives some clarification regarding why vesicles, in response to osmotic stress, reduce the amount neurotransmitters released during exocytosis release. The amperometric recordings here indicate this is a reversible process and that vesicle after osmotic shock are refilled with neurotransmitters when placed cells are reverted into an isotonic environment.

Osmotic stress affects exocytosis and the amount of neurotransmitter released during this process. We demonstrate how combining electrochemical methods together with transmission electron microscopy can be used to study the effect of extracellular osmotic pressure on exocytosis activity, vesicle quantal size, and the amount of neurotransmitter released during exocytosis.

Überwachung der Wirkung des osmotischen Druckes auf sekretorischen Vesikel und Exozytose

Amperometry recording of cells subjected to osmotic shock show that secretory cells respond to this physical stress by reducing the exocytosis activity ...

Rapid Isolation of Dorsal Root Ganglion Macrophages

There are growing interests to study the molecular and cellular interactions among immune cells and sensory neurons in the dorsal root ganglia after peripheral nerve injury. Peripheral monocytic cells, including macrophages, are known to respond to a tissue injury through phagocytosis, antigen presentation, and cytokine release. Emerging evidence has implicated the contribution of dorsal root ganglia macrophages to neuropathic pain development and axonal repair in the context of nerve injury. Rapidly phenotyping (or &#8220;rapid isolation of&#8221;) the response of dorsal root ganglia macrophages in the context of nerve injury is desired to identify the unknown neuroimmune factors. Here we demonstrate how our lab rapidly and effectively isolates macrophages from the dorsal root ganglia using an enzyme-free mechanical dissociation protocol. The samples are kept on ice throughout to limit cellular stress. This protocol is far less time consuming compared to the standard enzymatic protocol and has been routinely used for our Fluorescence-activated Cell Sorting analysis.

Here we present a mechanical dissociation protocol to rapidly isolate macrophages from the dorsal root ganglion for phenotyping and functional analysis.

Schnelle Isolierung von Dorsalwurzel Ganglion Makrophagen

There are growing interests to study the molecular and cellular interactions among immune cells and sensory neurons in the dorsal root ganglia after ...

Imaging Plasma Membrane Deformations With pTIRFM

To gain novel insights into the dynamics of exocytosis, our group focuses on the changes in lipid bilayer shape that must be precisely regulated during the fusion of vesicle and plasma membranes. These rapid and localized changes are achieved by dynamic interactions between lipids and specialized proteins that control membrane curvature. The absence of such interactions would not only have devastating consequences for vesicle fusion, but a host of other cellular functions that involve control of membrane shape. In recent years, the identity of a number of proteins with membrane-shaping properties has been determined. What remains missing is a roadmap of when, where, and how they act as fusion and content release progress.
Our understanding of the molecular events that enable membrane remodeling has historically been limited by a lack of analytical methods that are sensitive to membrane curvature or have the temporal resolution to track rapid changes. PTIRFM satisfies both of these criteria. We discuss how pTIRFM is implemented to visualize and interpret rapid, submicron changes in the orientation of chromaffin cell membranes during dense core vesicle (DCV) fusion. The chromaffin cells we use are isolated from bovine adrenal glands. The membrane is stained with a lipophilic carbocyanine dye,1,1&#39;-dioctadecyl-3,3,3&#39;,3&#39;-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate, or&#160;diD. DiD intercalates in the membrane plane with a &#34;fixed&#34; orientation and is therefore sensitive to the polarization of the evanescent field. The diD-stained cell membrane is sequentially excited with orthogonal polarizations of a 561&#160;nm laser (p-pol, s-pol). A 488&#160;nm laser is used to visualize vesicle constituents and time the moment of fusion. Exocytosis is triggered by locally perfusing cells with a depolarizing KCl solution. Analysis is performed offline using custom-written software to understand how diD emission intensity changes relate to fusion pore dilation.

Polarization-based Total Internal Reflection Fluorescence Microscopy (pTIRFM) enables real-time detection of cell membrane dynamics. This article describes the implementation of pTIRFM for the study of membrane remodeling during regulated exocytosis. The technique is generalizable to other processes in cell biology that directly or indirectly involve changes in membrane shape.

Imaging Plasmamembran Verformungen Mit pTIRFM

To gain novel insights into the dynamics of exocytosis, our group focuses on the changes in lipid bilayer shape that must be precisely regulated during ...

Biologie

Confocal Microscopy to Measure Three Modes of Fusion Pore Dynamics in Adrenal Chromaffin Cells

Dynamic fusion pore opening and closure mediate exocytosis and endocytosis and determine their kinetics. Here, it is demonstrated in detail how confocal microscopy was used in combination with patch-clamp recording to detect three fusion modes in primary culture bovine adrenal chromaffin cells. The three fusion modes include 1) close-fusion (also called kiss-and-run), involving fusion pore opening and closure, 2) stay-fusion, involving fusion pore opening and maintaining the opened pore, and 3) shrink-fusion, involving shrinkage of the fusion-generated &#937;-shape profile until it merges completely at the plasma membrane.
To detect these fusion modes, the plasma membrane was labeled by overexpressing mNeonGreen attached with the PH domain of phospholipase C &#948; (PH-mNG), which binds to phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) at the cytosol-facing leaflet of the plasma membrane; vesicles were loaded with the fluorescent false neurotransmitter FFN511 to detect vesicular content release; and Atto 655 was included in the bath solution to detect fusion pore closure. These three fluorescent probes were imaged simultaneously at ~20-90 ms per frame in live chromaffin cells to detect fusion pore opening, content release, fusion pore closure, and fusing vesicle size changes. The analysis method is described to distinguish three fusion modes from these fluorescence measurements. The method described here can, in principle, apply to many secretory cells beyond chromaffin cells.

This protocol describes a confocal imaging technique to detect three fusion modes in bovine adrenal chromaffin cells. These fusion modes include 1) close-fusion (also called kiss-and-run), involving fusion pore opening and closure, 2) stay-fusion, involving fusion pore opening and maintaining the opened pore, and 3) shrink-fusion, involving fused vesicle shrinkage.

Konfokale Mikroskopie zur Messung von drei Modi der Fusionsporendynamik in Nebennierenchromaffinzellen

Dynamic fusion pore opening and closure mediate exocytosis and endocytosis and determine their kinetics. Here, it is demonstrated in detail how confocal ...

Primary Culture of Rat Adrenocortical Cells and Assays of Steroidogenic Functions

The hormone which the adrenal cortex secretes is vital for animals against stress and diseases. The method described here is the procedure of primary cultured rat adrenal cells and related functional assays (immunofluorescence staining of lipid droplet surface protein, as well as corticosterone analysis). Unlike an in vivo model, the variation of interexperiments in adrenal monolayer cultures is less and the experimental condition is easy to control. Besides, the source of rats is also more stable than other animals, like bovine ones. There are also several human adrenal cell lines (NCI-H295, NCI-H295R, SW13, etc.) that can be used in adrenal studies. However, the steroid production of these lines will still be influenced by numerous factors, which include serum lot number, passage number, mutant/loss of distinct genes, etc. Except for lacking 17&#945;-hydroxylase, the primary culture of rat adrenocortical cells is a better and more convenient technique for studying adrenal physiology. In summary, primary rat adrenal cultures could be a good in vitro platform for researchers to investigate the mechanisms of the reagent of interest in the adrenal gland system.

The hormone secreted by the adrenal cortex is vital for animals against stress and diseases. Here, we present a protocol to culture the primary rat adrenal cells. It could be a good in vitro platform for investigating the mechanisms of the reagent of interest in adrenal steroidogenesis and lipid biosynthesis.

Primärkultur von Ratte Nebennierenrindeninsuffizienz Zellen und Assays Steroidogenic Funktionen

The hormone which the adrenal cortex secretes is vital for animals against stress and diseases. The method described here is the procedure of primary ...

Optimizing Isolation and Purification of Murine Glomerular Mesangial Cells

Mesangial cells (MCs) are stromal cells located in the middle space of the glomerulus, with pivotal functions in glomerular homeostasis. Methods for isolating, purifying, and culturing glomerular MCs have been developed and optimized since the 1980s for use in biomedical research, particularly in the field of nephrology. Mice are the most frequently used experimental animal models in research on renal diseases. In this study, we developed an optimized protocol for murine MCs isolation and ex vivo cell culture. These cells can be passaged multiple times, frozen, revived, and cultured without compromising cell growth or protein expression. This optimized approach significantly reduces the study duration for researchers and enables long-term cell preservation. The necessary equipment for the procedures is easily accessible in a basic biomedical laboratory, and the procedural steps are straightforward. The acquisition of target cells requires only 2-3 weeks, a reduction of at least 1 week compared to existing methods.

This study developed an optimized protocol for the isolation of murine mesangial cells (MCs) and their ex vivo cell culture. These cells can be passaged multiple times, frozen, revived, and cultured without compromising cell growth or protein expression.

Optimierung der Isolierung und Reinigung von murinen glomerulären Mesangialzellen

Mesangial cells (MCs) are stromal cells located in the middle space of the glomerulus, with pivotal functions in glomerular homeostasis. Methods for ...

Isolation of Macrophages from Mouse Dorsal Root Ganglion Using Mechanical Dissociation

Source: Yu, X., et al. <a href="https://app.jove.com/v/60023">Rapid Isolation of Dorsal Root Ganglion Macrophages.</a> J. Vis. Exp. (2019)
This video demonstrates an enzyme-free protocol to isolate macrophages from mouse dorsal root ganglion (DRG) using a mechanical dissociation technique. Upon homogenization of the freshly collected DRG tissue, antibodies against the cell-surface receptors on the macrophages are used to label the cells fluorescently, followed by performing flow cytometry to confirm the presence of macrophages in the sample.

Isolierung von Makrophagen aus dem Spinalganglion der Maus mittels mechanischer Dissoziation

Source: Yu, X., et al. Rapid Isolation of Dorsal Root Ganglion Macrophages. J. Vis. Exp. (2019)
This video demonstrates an enzyme-free protocol to isolate ...

JoVE Encyclopedia of Experiments

Immunology

Immune Systems and Components

Cell Isolation and Culture

Isolation of Microglia from the Cortex of an Adult Mouse Brain

Source: Zhou, L., et al., <a href="https://app.jove.com/v/60347">Isolation of Region-specific Microglia from One Adult Mouse Brain Hemisphere for Deep Single-cell RNA Sequencing.</a> J. Vis. Exp. (2019)
The video demonstrates a technique for isolating microglia from the cortex of an adult mouse brain. First, the cortex undergoes segmentation and homogenization to remove neurons and other glial cells, preserving microglia. Following that, the cell suspension is exposed to anti-myelin magnetic microbeads and a magnetic field, effectively separating microglia and myeloid cells from myelin debris and cells containing myelin.

Isolierung von Mikroglia aus der Hirnrinde eines adulten Mausgehirns

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Mechanical Tissue Dissociation
...