Macrophages: A Practical Approach (Practical Approach Series)
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The next two chapters cover measuring macrophage activity in vitro and in vivo. Finally methods are described for the analysis of gene expression in macrophages.
A variety of broad techniques have been brought together in one affordable volume to make Macrophage Methodology an essential buy for anyone studying macrophages. Convert currency. Add to Basket. Condition: New. Usually Dispatched within Business Days , Buy with confidence , excellent customer service. Seller Inventory More information about this seller Contact this seller.
Macrophages: A Practical Approach
Language: English. Brand new Book. Macrophage Methodology describes how to isolate moderate to high yields of viable cells from a variety of specific tissue sites under both normal and pathological conditions and then goes on to give protocols formacrophage purification. Chapter four identifies the key issues relating to the study of macrophages as antigen presenting cells and has protocols for the majorassays used to measure antigen processing and presentation.
Finally methods are described forthe analysis of gene expression in macrophages. Seller Inventory AAV New Book. Delivered from our UK warehouse in 4 to 14 business days. Established seller since Other Authors Paulnock, Donna M. Physical Description xvi, p. Series The practical approach series Practical approach series Subjects Macrophages. Macrophages -- Laboratory manuals. Macrophages -- Research -- Methodology. Immunologic Techniques. Contents Machine derived contents note: Isolation of macrophages from tissues, fluids, and immune response sites Purification of macrophages Characterization of macrophage antigens and receptors by immunochemistry and fluorescent analaysis Analysis of antigen processing and presentation Macrophage secretory products Analysis of macrophage lytic function Analysis of macrophage activity in vivo Analysis of gene expression in mononuclear phagocytes.
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Notes Includes bibliographical references and index. View online Borrow Buy Freely available Show 0 more links Set up My libraries How do I set up "My libraries"? Black Mountain Library. Monash University Library. Open to the public ; Since there are certain levels of lymphocyte contamination associated with isolation by these methods, cells can subsequently be further purified by adherence.
OptiPrep: 3 vol. OptiPrep: 4 vol. H20 and Adjust the pH to 7. Add Dilute this stock prior to use. Overlay the mixture with 10 ml of 1. Overlay the 1. Further, alveolar macrophages can be potent suppressors of T lymphocyte responses, which is important for the limitation of tissue damage 21 , These cells reside in an environment quite different than most macrophages due to the aerobic atmosphere. To collect human cells, the most common method is broncheolar lavagc 22 ; however, since willing subjects arc not always available, methods have been developed for cell isolation from lung biopsies One such procedure is outlined in Protocol 5.
Lung lavages are possible in rodents also 24, However, recovery is limited and in many cases, a more complicated procedure of mechanical and enzymatic dissociation is required 26, Place the lungs on a sterile Petri dish. A procedure for isolation of macrophages from whole murine lung is given in Protocol 7. Repeat the procedure three times. Do not leave the suspension on ice longer than 5 min. To enrich for the macrophage population, the cells from Protocol 7 can be passed through a Ficoll-Hypaque density gradient see Chapter 2.
The remaining cells are predominantly lymphocytes. By this method approximately 1 x macrophages can be obtained per animal Resident bone marrow macrophages are found within erythroid clusters and have a morphology and phenotype distinct from monocytes or bone marrowderived macrophages. In mice, each cluster contains an average of 35 cells.
The method described in Protocol 8 is based on that described by Crocker and Gordon 8, 28 and details the isolation of resident macrophages from the bone marrow of mice. In the isolation of resident bone marrow macrophages, it is important that any mechanical manipulations be very gentle. These cells have long plasma membrane processes which branch throughout the marrow stroma. Vigorous pipetting or passage through needles should be avoided as it will result in the loss of the mature macrophage population. Centrifugation should also be avoided to minimize artificial clustering of cells.
Large numbers of macrophages can be derived from bone marrow precursors. The isolation of these cells involves harvesting immature cells and culturing them with specific growth factors to continue haemalopoiesis in vitro Utilization of bone marrow-derived macrophages is desirable because a more homogeneous population of cells is obtained.
However, bone marrow-derived macrophages require about a week before they are ready to use, and due to the necessity of supplemental growth factors, can be a costly method. Approximately x bone marrow cells can be harvested per mouse which are then cultured under conditions which favour growth of the macrophage population 16, Protocol 9 describes the in vitro propagation of bone marrow-derived macrophages from pathogen-free mice. Resuspend the cells in 5 ml complete DMEM.
The yield of bone marrow-derived macrophages after seven days of culture should approach x macrophages per 10 x cultured bone marrow cells. Other growth factors such as granulocyte-macrophage colony-stimulating factor or interleukin-3 can be used in place of M-CSF.
Using these growth factors will result in a yield of 1 x macrophages per 10 x bone marrow cells Macrophages can be recovered from liver, gut, brain, bone, and spleen with varying degrees of time, purity, viability, and yield.
In other tissues, macrophage experimentation is mostly limited to histological techniques. To make single cell suspension of splenocytes. Isolation of human splenic macrophages requires a more rigorous protocol of tissue dissociation and is discussed in Protocol Remove the spleen by lifting it at one end with sterile forceps and cutting away from the body.
Place the spleen on sterile nylon mesh.
Macrophages: A Practical Approach
Press the spleen through the mesh with a sterile syringe plunger. LOPEZ or cloth screens. Unfortunately, this method can be overly rigorous. Over-mincing cells often leads to poor viability and it may favour survival of only hearty subpopulations of cells However, careful mechanical dissociation or a combination of mechanical and enzymatic manipulation may be the only method to obtain single cell suspensions of tough tissues.
When the quandtation of surface markers are desired, special care must be taken to be as gentle as possible with the tissues. In most enzymatic protocols a certain level of mechanical dissociation is necessary. The most widely used protease, i. Pronase and DNase also arc used in many protocols. One of the most important steps in any enzymatic protocol is to inactivate these enzymes after tissue dissociation, and to wash the resulting cell suspension thoroughly to remove all proteases The types and amount of enzyme used depends largely on the tissue being dissociated, and the desired cell type being isolated.
Macrophages Practical Approach by Paulnock Donna - AbeBooks
The following protocols were designed specifically for macrophage isolation from these tissue types, however, it is highly recommended that a few 'practice nans' he performed to optimize viability and purity. However, the human spleen is an abundant source of macrophages and these cells can be obtained by a combination of mechanical and enzymatic dissociation Macrophages can be further isolated by plastic adherence see Chapter 2 but for highly enriched cells without selective loss, countercurrent centrifugal elutriation is recommended see Chapter 2.
Kupffer cells The liver is comprised of parenchymal cells hepatocytes and non-parenchymal cells. The majority of non-parenchymal cells are sinusoidal cells. Hepatocytes can be separated from non-parenchymal cells due to their sensitivity to collagenase Approximately 1.