Isolation of Mature (Peritoneum-Derived) Mast Cells and Immature (Bone Marrow-Derived) Mast Cell Precursors from Mice.

Mast cells (MCs) are a versatile cell type playing key roles in tissue morphogenesis and host defence against bacteria and parasites. Furthermore, they can enhance immunological danger signals and are implicated in inflammatory disorders like fibrosis. This granulated cell type originates from the myeloid lineage and has similarities to basophilic granulocytes, both containing large quantities of histamine and heparin. Immature murine mast cells mature in their destination tissue and adopt either the connective tissue (CTMC) or mucosal (MMC) type. Some effector functions are executed by activation/degranulation of MCs which lead to secretion of a typical set of MC proteases (MCPT) and of the preformed or newly synthesized mediators from its granules into the local microenvironment. Due to the potential accumulation of mutations in key signalling pathway components of corresponding MC cell-lines, primary cultured MCs are an attractive mean to study general features of MC biology and aspects of MC functions relevant to human disease. Here, we describe a simple protocol for the simultaneous isolation of mature CTMC-like murine MCs from the peritoneum (PMCs) and immature MC precursors from the bone marrow (BM). The latter are differentiated in vitro to yield BM-derived MCs (BMMC). These cells display the typical morphological and phenotypic features of MCs, express the typical MC surface markers, and can be propagated and kept in culture for several weeks. The provided protocol allows simple amplification of large quantities of homogenous, non-transformed MCs from the peritoneum and bone marrow-derived mast cells for cell- and tissue-based biomedical research

Isolation of Hepatocytes from Liver Tissue by a Novel, Semi-Automated Perfusion Technology

Primary hepatocytes are a major tool in biomedical research. However, obtaining high yields of variable hepatocytes is technically challenging. Most protocols rely on the two-step collagenase perfusion protocol introduced by Per Ottar Seglen in 1976. In this procedure, the liver is perfused in situ with a recirculating, constant volume of calcium-free buffer, which is maintained at 37 °C and continuously oxygenated. In a second step, the liver is removed from the carcass and perfused with a collagenase solution in order to dissociate the extracellular matrix of the liver and liberate individual cells. Finally, the dissected hepatocytes are further purified and concentrated by density-based centrifugation. However, failure in proper cannulation, incomplete enzymatic digestion or over-digestion can result in low cell yield and viability. Here we present a novel semi-automated perfusion device, which allows gentle, rapid and efficient generation of a single-cell suspension from rodent livers. In combination with prefabricated buffers, the system allows reliable and highly reproducible isolation of primary hepatocytes

Ballooning of peritoneal cavity.

<p><b>(A)</b> A syringe is filled with PBS (3 mL) and air (2 mL) and the content <b>(B-E)</b> injected into the peritoneal cavity of the mouse. <b>(F)</b> This procedure results in a ballooned peritoneal cavity.</p

Schematic overview of the protocol for isolation of bone marrow cells.

<p>The hind legs are fixed, the skin of the legs is removed to the ankle, and the long bones of the legs of the fixed mouse are exposed by removing the muscle tissue (1). The legs are detached from the body at the hip joint and the ankle. The tibia and femur of each leg are separated at the knee joint (2), the bone ends are chopped off, and the bone marrow rinsed out with a syringe filled with BMMC medium (3) and collected in a Falcon tube (4). The cells are enriched by centrifugation (5) and the final cell pellet dissolved in BMMC medium (6) and added into Petri dishes filled with BMMC medium (7–8).</p

Extraction of peritoneal cell-derived mast cells.

<p>The mouse is carefully shaken in the palm of the operator, the air removed with a syringe and a small incision is made in the <i>linea alba</i> (not shown). <b>(A)</b> The PMC-containing solution is withdrawn from the peritoneal cavity using a Pasteur pipette and <b>(B)</b> placed into a sterile Falcon tube.</p

Schematic overview of the protocol for isolation of PMCs.

<p>Before starting with the isolation protocol, the mouse is anesthetized by inhalation of Isofluran and then sacrificed by cervical dislocation. In the first step of the isolation protocol, the abdominal skin is lifted up with forceps and an incision in the abdominal skin is made using scissors (1). The skin is fixed by forceps and torn towards the sternum, so that the cutis plus facia is separated from the lower fabric up to the top of the sternum to expose the abdominal wall (2). Using a 10-mL syringe that is filled with 3 mL of PBS and 2 mL of air (3), PBS and air is injected into the peritoneal cavity at the <i>linea alba</i> in direct proximity to the sternum (4–5). Thereafter, the mouse is carefully shaken in the palm of the operator (not shown). The mouse is then placed back on the operation table. Subsequently, the air that was brought in before is removed with a syringe (6). Subsequently, the cell-containing fluid of the peritoneal cavity is gently collected in a plastic Pasteur pipette (7) and placed into a Falcon tube (8). Cells are then pelleted by centrifugation (9). If the cell suspension shows a red colour due to contaminating blood cells, the resulting cell pellet is resuspended in Gey’s solution and left on ice for 5 min. For further purification, the cell suspension is underlain with fetal calf serum (10) and centrifuged (11). The final cell pellet is suspended in PMC medium (12) and plated into cell culture dishes (13).</p

Experimental set up for isolation of mast cells.

<p><b>(A)</b> A simple operational work area is prepared by wrapping an aluminium foil around a Styrofoam plate. All instrumentation that is necessary during the surgery is placed in clear order. <b>(B)</b> The sacrificed mouse is layered on the work area and the abdominal skin sterilized with 70% ethanol. <b>(C)</b> The abdominal skin is removed and the operation area (abdominal wall) exposed for further interventions.</p

Analysis of surface markers.

<p><b>(A)</b> PMCs and <b>(B)</b> BMMCs were stained for FcεRIα and Kit and analyzed by FACS. Side scatter (SSC) and forward scatter (FSC) in each sort are marked and the fraction of cells positive for Kit (<i>purple</i>), FcεRIα (<i>orange</i>) or both markers (<i>light blue</i>) indicated. Please note that in this sort, 97.1% of PMCs are positive for both markers, while only a small fraction of cells (2.8%) are positive for FcεRIα alone. In addition, 95.8% of all BMMCs were positive for both markers, while only 1.7% stained positive for Kit and 0.9% for FcεRIα alone.</p