Purification of macrophages by isopycnic gradient centrifugation
The density of a cell is a major physical characteristic, which can be used for purification and separation of cell populations by means of isopycnic centrifugation. It reflects the average chemical composition of a cell rather than surface characteristics or size. The various major cellular components all differ substantially in density. However, since most cells have roughly similar proportions of these components, the density range of cells is relatively narrow. In fact, most mammalian cells...
Preparation of in vitro macrophage cultures for immunochemical staining
Chapters 1 and 2 of this book cover the isolation and in vitro cultivation of macrophages. Section 1 contains a list of macrophage populations on which these techniques are commonly used. All human cells and serum must be treated as potentially contagious, with HIV or hepatitis especially, and must be handled and disposed of accordingly. When preparing isolated macrophages for immunochemical staining, we recommend plating them on glass covers lips. Protocol 7 describes one method for coverslip...
Preparation of fresh tissue for immunohistochemistry
Disposable moulds Raymond A. Lamb Tissue-Tek OCT compound Bayer Diagnostics 1 Before dissection, place a small amount of iso-pentane in a small Pyrex dish. 2 Place the dish in a polystyrene box containing a layer of 'dry ice' C02 . Freshly obtained mouse or human tissue 3 Cover box to lower temperature quickly. 4 Fill a mould with Tissue-Tek, ensuring as few air bubbles as possible. 5 Place the tissue carefully in mould.c excluding air bubbles, and orientating it so that the required plane...
Assessment of macrophage antiLeishmania cytolytic activity
Assorted sterile plastic pipettes, conical tubes, tissue culture dishes, and other labware see Protocol 1 Complete media RPMI 1640 medium Gibco BRL supplemented with 0 FCS HyClone , 100 U ml penicillin, 100 M-g ml streptomycin, 2 mM L-glutamine, 20 mM Hepes Gibco BRL . for macrophages 199 medium supplemented with 20 heat-inactivated FCS HyClone . 100 U ml penicillin, 100 g ml streptomycin, 2 mM L-glutamine, 40 mM Hepes, 0.1 mM adenine in 50 mM Hepes . hemin 5 ig ml in 50 tri ethano amine , and...
Antifungal activity
Candida albicans is an opportunistic pathogen whose relevance has increased in recent years because of the augmented incidence of candidiasis in immunocompromised hosts. C. albicans can be considered a facultative intracellular pathogen because it survives within the macrophage and grows out of this cell by germination into a highly pathogenic form, C. albicans can undergo dimorphic-transition in vitro and in vivo from the yeast Y-Candida to the hyphal U-Candida form. The anti-Candida activity...
Equipment and reagents Rjh
Sterile and nuclease-free microcentrifuge tubes 1,5-2.0 ml, Life Science Products, Inc. Sterile and nuclease-free micropipette tips, with aerosol barrier Molecular Bio-Products, Inc. Microcentrifuge Dupont NEN Macrophage population of interest see Chapters 1 and 2 for details RNA STAT-60 extraction buffer Tel-Test 'B Inc. DEPC-treated H20 add 1 ml of diethyl pyrocarbonate DEPC, Sigma Chemical Co. per litre of water, stir for 1 h at room temperature, and autoclave for 1 h to hydrolyse any...
Equipment and reagents Php
MICROTEST tissue culture plates. 96-well, flat-bottom, polystyrene Falcon Plastics, Becton Dickinson Labware Device for vacuum aspiration Combitips 1-5 ml Eppendorf Complete medium RPMI1540 see Protocol 6 Triton X-100 Sigma Chemical Sabouraud dextrose agar Biolife Monocyte macrophage effector cells, appropriately purified see Chapters 1 and 2 Macrophage activators see Protocol 1
Measurement of macrophage cytotoxicity using the indium release assay
Tissue culture plates, 96-well, U-bottom, polystyrene Corning-Costar Assorted sterile plastic pipettes, conical tubes, tissue culture dishes, and other labware see Protocol i Device for vacuum aspiration n,In-oxyquinoline nllnOx , specific activity 50 mCi fig In Amersham Life Science Combttips Eppendorf Complete medium see Protocol 1 Ca2 -, Mg -free Dulbecco's modified PBS see Protocol 1 0.05 w v trypsin, 0,02 EDTA solution Hyclone Laboratories Macrophage effector cells, appropriately purified...
A Preparation of effector cells
1 Resuspend monocytes macrophages at 2 x 10s cells ml in complete medium. Complete medium see Protocol J Ca2 -, Mg21 -free Dulbecco's modified PBS see Protocol 1 0.0556 w v trypsin, 0.02 EDTA solution Hyclone Laboratories Macrophage effector cells, appropriately purified see Chapters 1 and 2 Tumour target cells e.g. the P815 murine mastocytoma cell line, maintained in complete medium by serial passages on 100 mm tissue culture dish or in 75 cm2 tissue culture flasks Macrophage activators see...
AntiLeishmania activity of monocytesmacrophages
Intracellular survival and proliferation are primary mechanisms adopted by many infectious agents for evading the immune response of their vertebrate host. Protozoa of the genus Leishmania reside and multiply in the macrophage phagolysosomes. Leishmaniae exhibit a heteroxenous life cycle that includes two developmental stages an extracellular, flagellated leptomonad form promasti-gote and a sessile form amastigote which is an obligate intracellular parasite. Promastigotes are taken up by...
B Determination of clodronate concentration
1 Prepare a standard curve using 0,10, 20,40, 50, 70, and 80 p.1 of the extracted standard clodronate solution added with saline to a total volume of 1 ml per tube. 2 Dilute the samples until they are within the range of the standard curved 3 Add 2.25 ml of 4 mM CuSO solution, 2.20 ml Milli Q, and 0.05 ml HNO, solution to each tube, containing 1 ml sample or standard. 5 Read the samples at 240 nm using a spectrophotometer. a Storage at -20 C. When pipetting phenol with glass pipette, be sure...
nuclear runon assay
Nucleotide bases 100 mM in lithium salt solution Boehringer Mannheim 10 x SET buffer combine appropriate volumes of 20 SDS, 0.5 M EDTA, 1 M Tris pH 7.8 to give a final concentration of 10 SDS. 50 mM EDTA, and 100 mM Tris pH 7.4 use autoclaved solutions and autoclaved bottle for preparation do not autoclave final solution Proteinase K solution 20 mg ml in 1 x SET buffer store at -20 C PCt phenol, chloroform, isoamyl alcohol solution first combine 24 parts chloroform and one part isoamyl...
Isolation of whole mononuclear cells from peripheral blood by the FicollHypaque
This simple and rapid method takes advantage of the density differences between mononuclear cells and other elements found in blood samples. Mononuclear cells and platelets collect on top of the Ficoll-Hypaque layer as they have a lower density. In contrast, red blood cells and granulocytes have a higher density than Ficoll-Hypaque and collect at the bottom of Ficoll-Hypaque layer. Platelets are separated from the mononuclear cells by subsequent washing or centrifugation through a FCS cushion...
Onestep continuous Percoii gradient separation of monocytes
PBMC purified by Ficoll-Hypaque gradient Protocol 8 Ca Mg -free PBS see Protocol 1 RPMI1640 medium supplemented with 15 FBS Iso-osmotic Percoll. To prepare an osmotically balanced stock solution of Percoll Pharmacia mix 9.25 parts of concentrated Percoll with 0.75 parts v v of 10 x Ca2 Mg2 -free PBS. Adjust the osmolality of the Percoll solution, as well as of the RPMI 1640 medium supplemented with 15 FBS, to 285 mOs with 10 x Ca2 Mg2H -free PBS or distilled water, respectively. 1 Prepare a 46...
C Cytotoxicity assay
1 Add 1 x 104 pre-labelled target cells in 0,2 ml of complete medium into each well of a 96-well plate containing 2 X 105 effector cells E T cell ratio of 20 1 . Labelled tumour cells should also be added to wells without macrophages to determine the spontaneous release release by tumour cells alone and the total incorporated counts, 5 2 After 48-72 h in culture, harvest 0,1 ml of supernatant from each well with an automatic pipette. 3 When 3H TdR is used, place supernatants in vials containing...
Preparation of periodatelysine paraformaldehyde for perfusion of mouse tissue3
Lysine monohydrochloride Sigma dissolve 6.85 g in 187.5 ml dH20 - Sorensen's salt Na2HP0 .2H20 Sigma dissolve 0.9 g in 50 ml dH20 Paraformaldehyde BDH dissolve 10 g of paraformaldehyde in 100 ml dH20 and ten drops of 1 M NaOH at 56 C, then cool to room temperature 0.1 M phosphate buffer 7,8 g NaH2PO .2HiO BDH dissolved in 250 ml dHjO and 34 g Na2HP04.2H20 BDH dissolved in 1 litre dH,0. Mix the dissolved NaH2P0 .2H20 and Na2HP04.2Hi0 and dilute 1 1 to make the 0.1 M working solution. 1 Buffer...
Sources and characteristics of binding factors
The processes controlling gene transcription have been defined in substantial detail and generally involve the activity of a broad collection of proteins, many of which function through direct interaction with DNA 11 . There are many DNA binding factors which are constitutively active and are involved either in the regulation of constitutively transcribed genes or are part of general transcription machinery of the cell. With regard to inducible gene expression, however, many transcription...
Macrophage heterogeneity
It has long been recognized that macrophages isolated from different anatomical sites are heterogeneous with respect to many different properties including size, morphology, density, expression of cell surface proteins, and functional capabilities reviewed in refs 12, 70 . Because macrophage function is dependent, at least in part, on signals received from the immediate microenvironment, it has been suggested that macrophage heterogeneity may arise from the unique conditions within specific...
Contents
Preface page v List of protocols xi Abbreviations xv 1 Isolation of macrophages from tissues, fluids, and immune response sites 1 Mary Ellen Handel-Fernandez and Diana M. Lopez 2 The heterogeneity of macrophages 1 3 Isolation of free mononuclear phagocytes 2 Murine peritoneal cells 2 Blood monocytes 6 Alveolar macrophages 7 4 Macrophages in haematopoietic tissues 10 Mechanical and enzymatic digestion of tissue 15 6 Macrophages in immune response sites 25 Macrophages in infection 25...
Quantitation of macrophage endocytic function
Titertek 96-well plate fluoroplate Flow Laboratories Fluorimetric plate reader Fluoroscan 11, Labsystems Endocytic tracers see Table 3 labelled with fluorochromes see Protocol 9 Detachment buffer see Protocol 2 1 Triton X-100 Sigma in 10 mMTris buffer diluted from a 1 M stock dissolve 60,57 g of Tris base Sigma in 400 ml H20 adjust the pH to 7.5 using conc. HC1 4 paraformaldehyde see Protocol 3 BCA Protein Assay Reagent Kit Pierce Chemical Co. Adherent macrophages as a monolayer in 24-well...
by EDTA treatment
1 1 mixture of 10 mM EDTA in PBS pH 7.4 and RPMI 1640 medium containing 20 FBS mixture A 1 Prepare a monolayer of adherent monocytes as described in Protocol 1. 2 Collect the non-adherent cell fraction as described in Protocol 5, step 2. For 75 cm2 flasks, add 10 ml of mixture A. Incubate cells for 15 min at 37 C. 3 Collect the detached cells generally more than 90 by gentle shaking of the flask and pipetting, 4 Transfer the cell suspension to 50 ml conical centrifuge tubes, 5 Centrifuge at...
Cytokines and chemokines chemotaxis
Most cytokines have been identified thanks to appropriate bioassays, which have also provided tools to measure these mediators and to standardize them 3 . Even when immunometric methods are available, bioassays are invaluable tools to assess the functional relevance of immunoreactive material and to quantitate undefined mediators. Here we will focus on chemotaxis, the eponimous function of the chemokine superfamily 4, 5 . N-terminal processing of chemokines results in products with reduced...
Isolation of alveolar macrophages from whole murine lung
Sterile wire mesh screens PGC Scientific Sterile forceps, scissors, and scalpel 15 ml conical centrifuge tube RPMI 1640 containing 5 PCS Gibco BRL Dissociation medium RPMI 1640, 5 PCS. collagenase type I 150 U ml Sigma Chemical HBSS Gibco BRL see Protocol 5 DNase 150 U ml Sigma Chemical 1 Sacrifice the mouse by cervical dislocation and remove the lungs. 2 Pass the lungs through a Petri dish containing HBSS to eliminate contaminating blood, 3 Mince the lung tissue into 1 mmJ pieces with a...
Abbreviations
ADCC antibody-dependent cellular cytotoxicity CCE counterflow centrifugal elutriation DMEM Dulbecco's modified Eagle medium EDTA ethylenediamine tetraacetic acid EMSA electrophoretic mobility shift assay FITC fluorescein isothiocyanate GBSS Gey's balanced salt solution GM-CSF granulocyte-macrophage colony-stimulating factor HBSS Hank's balanced salt solution sulfonic acid MHC major histocompatibility complex PBMC peripheral blood mononuclear cells PDGF platelet-derived growth factor RNI...
Soluble cytokine receptors
Soluble receptors leave cells producing them and can be active in the cellular microenvironment as well as in body fluids 17, 18 . The discovery that membrane bound receptors are also released in body fluids has dramatically changed our understanding of the ligand-receptor interactions as well as, more in general, of the mode of action of hormones and cytokines. Ligand concentrations can be modified by soluble receptors, by down-regulation of the number of membrane bound receptors which are...
Selectivity of the approach with respect to macrophages
Since the liposome-mediated macrophage suicide technique is based on a rapid internalization and consecutive intracellular degradation of liposomes, it is not surprising that macrophages are the only cells to be affected. Indeed the approach allows the selective removal of mononuclear phagocytes from heterogeneous spleen cell populations in vitro. No effect was found on non-phagocytic spleen cells as measured by growth, protein production, antigen presentation, and antigen-specific T cell...
Isolation of macrophages from dissociation
Sterile 250 ji.m nylon mesh screens PGC2 Scientific Sterile forceps and scissors 75 mm2 sterile Petri dishes 19 Nycodenz density 1.1 g ml Accurate Chemical Co see Protocol 13 1 Cut the tumour into 2-3 mm3 pieces of tissue. 2 Pass the tissue pieces through a sterile screen mesh into a Petri dish by pressing on tumour sections with a sterile rubber stopper. 3 Collect the cell suspension in a fresh 75 mm2 Petri dish containing 2-3 ml of ice-cold RPMI1640 with 10 FCS. 4 Layer the resulting cell...
Induction of MHC class II antigens on bone marrow macrophages
4 ml polypropylene culture tubes Falcon 2063 '6 2' bone marrow-derived macrophage BMM0, see Protocol 1 Complete tissue culture medium supplemented DMEM see Protocol i with 10 FCS Recombinant murine IFN-y Genzyme 1 Adjust BMM0 to 106 ml in complete tissue culture medium. 2 Aliquot 3 x 106 per tube to the required number of polypropylene culture tubes. 3 Add recombinant murine IFN-y to a final concentration of 100 U mLb 4 Incubate cells for 48 h at 37 C, 5 C02. 5 Resuspend BMM0 by gently...
Isolation of murine Kupffer cells
150 cm2 tissue culture flask 15 inland 50 ml polypropylene tubes 24-gauge cannula Popper and Sons Sterile scissors and forceps Sterile steel mesh PGC Scientific Sterile 75 m sterile nylon mesh PCG Scientific 1 Sacrifice the mice by cervical dislocation or C02 inhalation. 2 Pin the mice to a dissecting board, abdomen up. 3 Clean the mouse with 70 ethanol. 4 With sterile scissors, make a ventral midline incision exposing the peritoneal cavity. 5 To perfuse the liver, first cut the vena cava to...
Problems caused by LPS contamination during the course of macrophage
LPS is a major factor in the modulation of macrophage functions and is capable of affecting the macrophage physiology even at very low concentrations reviewed in ref. 93 . LPS contamination during purification steps may result in marked effects on the adhesion properties of these cells. For example, it has been shown that the presence of low levels of LPS results in the recovery of weakly adherent subpopulations exhibiting a high cytolytic capacity 94 . Low concentrations of LPS also may result...
Modified protocols for use with pathogens
As previously stated, each pathogen may bring with it its own set of specific requirements. These often become most apparent when a pathogen is transfected to express a normally soluble 'reporter' antigen e.g. ovalbumin and then processing of the same epitopes are compared after these two modes of delivery 17, 19 . Soluble protein antigens can be readily titrated into APC assays within a concentration range from nanogram to milligram, normally without discernible adverse effects. Such...
Isolation of human blood monocytes
10 ml EDTA K3 Vacutainers Beckton Dickinson Hepes-buffered saline HBS 0,8 w v NaCl, 10 mM Hepes-NaOH pH 7.4 filter sterilized OptiPrep Accurate Chemical Co. Solution A HBS, 10 mM EDTA filter sterilized Solution B 0.5 w v BSA in solution A prepare fresh and filter sterilize OptiPrep 1,078 g ml 1 vol. OptiPrep 3 vol. solution B OptiPrep 1.068 g ml l vol. OptiPrep 4 vol. solution B Phosphate-buffered saline PBS 0.01 M phosphate, 0.15 M NaCl. Dissolve 20.5 g NaH2P04.Hj0 and 179.9 g NaHP0 -7H20 in...
General considerations on the adherencebased methods
Although all of the protocols outlined above can be used successfully, it must be acknowledged that techniques based upon the ability of monocytes macrophages to adhere to different surfaces may exhibit several disadvantages. They include poor cell viability, low yield, and contamination of cell preparations with other cell types. Based on the assumption that macrophages are not the only cell types capable of adhering to plastic or glass surfaces, adherence techniques alone cannot yield a 100...
Indirect immunofluorescent staining of cultured macrophages
Flow cytometer PBS see Protocol 2 4 paraformaldehyde see Protocol 3 Saponin Sigma or Triton X-100 Sigma Blocking buffer 5110 normal serum of the Primary antibody species of secondary antibody diluted in . Labelled secondary antibody see Section PBS 2.2.4 1 Detach1' adherent cells from the culture dishes see Protocol 2 f and place in appropriate size tube. 2 Harvest the cells by centrifugation at 1500gfor 5 min. 3 Resuspend the cell pellet in fresh 4 paraformaldehyde and leave on ice for 10...
Flow cytometry
3.1.1 Applications of flow cytometry How cytometry can measure the size and granularity of cells from their light scattering properties, in addition to determining the expression of antigenic markers by fluorescence detection. Flow cytometry may also be used to measure the relative uptake of fluorescent endocytic and phagocytic tracers see Sections 5.2 and 5.3 . Macrophages can be distinguished and sorted from other cell types in a complex cell mixture on the basis of size and granularity,...
B Cytotoxicity assay
1 Add 5 x 103 labelled target cells in 0.2 ml of complete medium to triplicate wells containing 2 x io5 macrophage effector cell to give a 40 1 E T cell ratio. 2 Incubate the cells in a humidified atmosphere containing 10 C02 at 37 C. The cytolytic activity of human monocytes is measured in a 48 h lnIn release assay, whereas killing by murine macrophages in a 24 h assay. 3 At the end of the incubation time, centrifuge the plates at 250-300 g for 5 min. 4 Remove 0.1 ml of supernatant fluid from...
Purification of macrophages by adherence to collagencoated surfaces
RPMI 1640 medium alone and containing 15 heat-inactivated FBS A Preparation of collagen-coated matrices 1 Dissolve lyophilized type I collagen at 1.5 mg ml in 0.1 M HOAc at 4 C by stirring overnight. 2 Dispense 250 of type I collagen solution to each well of 24-well cluster plates. 3 Simultaneously, add 25 jl1 of 10 x culture medium and 15 xl of 0.142 M NaOH to each well to bring the pH of the mixture to 7.6, 4 Incubate plates for 1-2 h at 37 C gelation usually occurs in 30-60 min , then wash...
Purification of macrophages by adherencebased methods
2.1 Adhesion properties of macrophages Macrophages are large cells which attach tenaciously to solid substrates during short periods of in vitro culture. The adherence of macrophages to solid substrates is an energy- and pH-dependent process that is enhanced by high serum concentration and low pH 16 . This property of mononuclear phagocytes has been used extensively to deplete lymphoid cell suspensions of monocytes macrophages and to prepare macrophage cultures from different anatomic sites....
Method Jel
1 Wash recipient macrophages twice at room temperature in a large volume of serum-free RPMI1540 medium by centrifugation for 5-10 min each at 250-500 g. 2 Remove the supernatant fluid from the final cell pellet by aspiration. 3 Resuspend the macrophages in cold PBS to a final concentration of 3 x 106 cells ml or higher. 4 Transfer 300 jxl of this cell suspension into an electroporation cuvette, keeping everything on ice. 5 Add plasmid DNA to the macrophage suspension to a final DNA...
Fixed tissue macrophages
Macrophage subpopulations located in different tissues are interesting to study since they are adapted to specialized activity within their local environment. Because they are closely associated with surrounding tissues, mechanical and or enzymatic digestion is often necessary. 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....
Introduction to the endocytic pathway
Macrophages are able to ingest particulate and soluble matter efficiently by phagocytosis and endocytosis. These processes are essential for macrophage antigen presentation and removal of pathogens. Below is a brief introduction to some of the components of the endocytic pathway, for a comprehensive review see refs 12 and 13. Endocytosis includes pinocytosis, receptor-mediated endocytosis, and phagocytosis. Pinocytosis is the non-specific sampling of the extracellular milieu and occurs by the...
Preparation of 4 paraformaldehyde
0.22 nm filter BDH 5 M NaOH Paraformaldehyde BDH PBS see Protocol 2 1 Dissolve 4 g of paraformaldehyde in 90 ml PBS containing one to two drops of 5 M NaOH by mixing at 56 C. 2 Make the solution up to 100 ml by adding 10 ml of 1 M Hepes. 3 Check the pH to ensure that it is approximately 7, 4 Filter the paraformaldehyde using a 0.22 j.m filter. 5 Chill to room temperature or 4 C, as appropriate, before use and use immediately.a a The paraformaldehyde should be made fresh. It also can be...
Injection of liposomes and access to tissue macrophages
A suspension of clodronate-liposomes prepared according to Protocol 1 contains about 6 mg clodronate per 1 ml suspension. Splenic macrophages can be depleted by intravenous administration of about 0.1 ml of a suspension of clodronate-liposomes prepared according to Pro toco 1 per 10 g body weight. Macrophages in the liver Kupffcr cclls arc more susceptible and can be completely depleted by intravenous administration of 0.02 ml of the suspension per 10 g body weight. See the relevant literature...
Detaching cultured macrophages from plastic and glass surfaces using
Phosphate-buffered saline PBS Sigma Polypropylene tubes various sizes combine 8.0 g NaCl, 0.2 g KH2PO 2.9 g . Cultured macrophages prepared as Na,HP04.12H20. 0.2 g KC1 all Sigma in 1 described in Chapter 2 in flasks or tissue litre dH30, and pH to 7.3 culture dishes Detachment buffer 0 combine 10-15 mM Ltdocaine-HCl Sigma and 10 mM EDTA in PBS 1 Aspirate the culture medium from the cells. 2 Wash the monolayers twice with PBS. 3 Add the detachment buffer. Use approx. 10 ml buffer per 100 cm2 of...
Practical considerations for immunofluorescent staining of macrophage
2.2.1 Direct versus indirect immunochemical labelling There are two methods to detect antigens expressed by macrophages 5 . Coupling of a detectable label fluorochrome or enzyme to an antigen-specific antibody Ab can be used for direct identification of the corresponding antigen. Indirect immunochemical staining utilizes an unlabelled primaiy Ab which is detected by a labelled secondary Ab raised against IgG of the species that produced the primary reagent. Background labelling, caused by...
List of suppliers
Accurate Chemical, 300 Shames Drive, Westbury, NY 11590, USA. Affinity Bloreagents Inc., Cambridge Research Biochemicals, Gadbrook Park, Northwich, Cheshire CW9 7RA, UK. Affinity Bioreagents Inc., 14818 West 6th Avenue, Suite 13A, Golden, CO 80401, USA. American Diagnostics inc., PO Box 1165, Greenwich, CT 06836-1165, USA. Anderman and Co. Ltd., 145 London Road, Kingston-upon-Thames, Surrey KT2 6NH, UK. Tel 0181 541 0035 Fax 0181 541 0623 Bayer Diagnostics, Strawberry Hill, Newbury, Berkshire,...
Ellcitation of peritoneal macrophages using thioglycollate
23-gauge needles and 5 ml syringes Brewer's thioglycollate medium. To prepare this weigh out 30 g of dehydrated thioglycollate medium and suspend in 1 litre of distilled water in a 2 litre Erhlenmeyer flask. Heat the thioglycollate solution to boil over a flame. Carefully 1 Clean the abdomen of each mouse with 70 ethanol. 2 Draw 2-3 ml of thioglycollate medium into a 5 ml syringe, attach a 23-gauge needle and inject the solution i.p. A large gauge needle is recommended because of the viscosity...
Isolation of human splenic macrophages
Tenbroeck tissue homogenizer Bellco Sterile gauze 2 in x 2 in Fisher Scientific Sterile forceps and scissors MP medium RPMI 1640. 2 M l-glutamine, 10 ng ml garamycin, 1 tiypticase soy broth, 10 heat-inactivated newborn calf serum Gibco BRL 1 Cut the splenic tissue 10-20 g into pieces in a Petri dish containing 5-10 ml MP medium. 2 Load the contents of the Petn dish into a Tenbroeck tissue homogenizer and homogenize the sample with eight to ten strokes. Collagenase type VIII Sigma Chemical Co...
Nonadherent versus adherent culture of macrophages
In the light of the well-known effect of adherence in macrophage activation, attention should be paid to defining the culture conditions of cells isolated by physical methods. As described in Section 2, if activation functions of macrophages are to be studied, it is advantageous to perform these studies on nonadherent macrophages. A variety of non-adherent systems have been established using monocytes isolated by centrifugal elutriation or density gradient centri-fugation. The use of culture...
Peritoneal macrophage elicitation using BioGel polyacrylamide beads
18-gauge needle, 5 ml syringe 75 xm sterile mesh screens PGC Scientific swirl the solution, dissolving the medium completely. The colour will change from brown to red. Take the solution off the flame after it begins to boil. Aliquot the thioglycollate into 100 ml or 250 ml bottles, and autoclave at 15 lb in2,121 C for 15 min, slow exhaust. Store in the dark at room temperature for one to two months before use.3 Bio-Gel P-100 fine beads Bio-Rad Laboratories RPMI 1640 with 10 FCS Gibco BRL 1...