Mouse Model for Cytomegalovirus Infection

Cytomegaloviruses (CMVs) are prototypes of the P subgroup of herpesviruses. They possess large DNA genomes of 230 kb with more than 200 open reading frames (Mocarski, 1996). Thus, they range among the mammalian viruses with the largest coding capacity. Although a high degree of species specificity is characteristic for cytomegaloviruses, viruses with a similar genomic structure and pathobiology are found in virtually all mammalian species analyzed. In vivo, CMVs infect a broad range of cells...

Induction of Inflammatory Bowel Disease in Immunodeficient Mice by Depletion of Regulatory T Cells

The severe combined immunodeficiency (SCID) model of colitis shares many features of idiopathic inflammatory bowel disease (IBD) in humans, some forms of which are thought to involve differential activation of TH1 cells (Breese et al., 1993). The SCID model is highly reproducible and easily manipulated, and as such provides a useful tool for studying mucosal immune regulation as it relates to the pathogenesis and treatment of IBD in humans. This unit describes a cell transfer system in which...

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Figure 20.2.3 In vivo characteristics of primary 4T1 tumors. (A) 4T1 tumors are highly vascularized. (B) Ulcerations of the skin can appear where primary 4T1 tumors are growing. (C) 4T1 tumors can extend to the peritoneal lining. Figure 20.2.3 In vivo characteristics of primary 4T1 tumors. (A) 4T1 tumors are highly vascularized. (B) Ulcerations of the skin can appear where primary 4T1 tumors are growing. (C) 4T1 tumors can extend to the peritoneal lining. mm in diameter. These data indicate...

Dialyze antibody and activate HRPO

Using a 15,000 MWCO dialysis membrane, dialyze 5.0 mg of antibody to be conjugated overnight at 4 C against 1 liter of 50 mM NaHCO3, pH 8.0, changing the solution once. 2. Add 5.0 mg HRPO to 1.5 ml distilled water in a 10-ml glass flask. Incubate 30 min at room temperature. 3. Add 0.25 ml of 0.1 M sodium periodate. Incubate 20 min at room temperature. 4. Add 0.25 ml of 0.5 M ethylene glycol to quench sodium periodate. Incubate 10 to 20 min at room temperature.

Mouse 4T1 Breast Tumor Model

The 4T1 mammary carcinoma is a transplantable tumor cell line originally isolated by Fred Miller and colleagues (Dexter et al., 1978 Aslakson and Miller, 1992). The tumor grows in BALB c mice and in tissue culture. It is one of four sublines derived from the 410.4 tumor, which was isolated from a single spontaneously arising mammary tumor of a MMTV+ BALB c mouse foster nursed on a C3H mother (BALB BfC3H Dexter, et. al., 1978). 4T1 was selected without mutagenesis for its resistance to...

Mouse Model Of Multiple Pulmonary Melanoma Metastases

The pulmonary metastasis model is the other widely used model for the evaluation of therapy in many tumor models, including B16 melanoma. Since essentially all tumor cells that take upon intravenous tumor injection are found in the lungs, the term pulmonary metastasis is widely used even though every resulting pulmonary nodule is technically an independent primary tumor rather than a true metastasis. The typical dose used is 2 x 105 cells mouse, which will yield between 50 and 250 pulmonary...

Induction Of Iddm In Diabetesresistant Bb Rats

Diabetes-resistant (DR)-BB rats with induced diabetes offer advantages over spontaneously diabetic diabetes-prone (DP)-BB animals. Untreated DR rats are not lymphopenic and their T cell populations are normal with respect to phenotype and function. Treatments to induce diabetes do not induce severe lymphopenia. In addition, T cells from diabetic DR-BB rats adoptively transfer IDDM with no requirement for mitogen activation (see Alternate Protocol 2). Another practical advantage is that cells...

The P815 Mastocytoma Tumor Model

P815 is a mastocytoma cell line derived by methylcholanthrene treatment of a male DBA 2 mouse. It is commonly used as an experimental tumor model not by virtue of its mast cell lineage (mastocytomas are clinically rare tumors) but because it offers several advantages for in vivo experimentation. Wild-type P815 cells grow progressively in the majority of syngeneic DBA 2 mice. They can be implanted either intraperitoneally (see Basic Protocol 1), which allows for colony assays to be performed to...

Tumor Protection Using Genetically Modified Whole Tumor Cell Vaccines Granulocytemacrophage Colonystimulating

Additional lymphoma antigens have not been defined. Therefore, one general approach to active immunotherapy is to utilize genetically modified autologous tumor cells as a source of Ag. Expression of various cytokine genes in tumor cells may enhance the immunogenicity and potency of cell-based vaccines. Tumor cells are modified transduced in vitro to express immuno- modulatory molecules such as cytokines (GM-CSF, IL-2, and IL-4), chemokines (IP-10), or costimulatory molecules (B7.1 and B7.2)....

Diagnosis Of Insulindependent Diabetes Mellitus Iddm

Monitoring of glycemic status should begin when NOD mice reach 10 weeks of age. Generally, this is done at weekly intervals by using Diastix (Bayer Diagnostics appendix 5) or similar reagent strips to measure urine glucose. Picking a mouse up leads to immediate urination, allowing a drop to be collected on the test area (tip) of the reagent strip. High levels of glucose in the urine (glycosuria) appear when plasma glucose is > 300 mg dl. A nonfasting plasma glucose of > 300 mg dl for 2...

Selection and Preparation of Dialysis Membrane

Dialysis membranes are available in a number of thicknesses and pore sizes. Thicker membranes are tougher, but restrict solute flow and reach equilibrium more slowly. Pore size is defined by molecular weight cutoff (MWCO) i.e., the size of the smallest particle that cannot penetrate the membrane. The MWCO designation should be used only as a very rough guide if accurate MWCO information is required, it should be determined empirically (see Craig, 1967, for a discussion of parameters affecting...

Types Of Detergents

A large variety of detergents are available (Helenius et al., 1979). For biochemical studies, they are usually categorized according to the type of hydrophilic group anionic, cat-ionic, amphoteric, or nonionic. Tables A.1D.1 and A.1D.2 list commonly used members of each type. In general, nonionic and amphoteric detergents are less denaturing for proteins than ionic detergents. Sodium cholate and sodium deoxycholate are the least denaturing of the commonly used ionic detergents. Two properties...

Syngeneic Trampc1 And C2 Subcutaneous Tumor Model

Previously, two prostate cancer cell lines, TRAMP-C1 and -C2, were established from a tumor harvested from a 32-week-old C57BL 6 TRAMP male (Foster et al., 1997). These lines retain expression of androgen receptor (AR), E-cadherin, and cytokeratin supporting their tissue-specific origin from prostate epithelial cells following malignant transformation. Interestingly, unlike autochthonous TRAMP tumor cells, these lines no longer express TAg (Foster et al., 1997 Kwon et al., 1997). Nevertheless,...

B16 as a Mouse Model for Human Melanoma

Malignant melanoma is the sixth most common cancer in the U.S., with an estimated 44,200 new cases reported in 1999 (Centers for Disease Control and Prevention, 1999). A subset of patients with metastatic melanoma can be successfully treated by the administration of recombinant interleukin-2 (rIL-2), sometimes given together with autologous melanoma-reactive lymphocytes that have been expanded ex vivo (Rosenberg, 1997 Rosenberg, 1999). Recently, a number of different laboratories have used...

Induction Of B16 Melanoma Protection By Recombinant Vaccinia Virus rVv Vaccine

Currently, the two tumor vaccines that induce the most reliable protection of mice from lethal B16 challenge are rVV encoding the MDA, mTRP-1 and irradiated B16 expressing GM-CSF (Hung et al., 1998 Overwijk et al., 1999). Although immunization with rVVhgp100 induces high levels of gp100-specific CTL, this regimen is completely ineffective in preventing B16 growth upon intravenous or subcutaneous challenge. However, adoptive transfer of in vitro cultured, gp100-reactive CTL can greatly reduce...

Induction Of Experimental Autoimmune Thyroiditis Using Cfa As Adjuvant

Experimental autoimmune thyroiditis (EAT) can be induced by injecting mice with mouse thyroglobulin (MTg) emulsified in complete Freund's adjuvant (CFA) supplemented with Mycobacterium tuberculosis H37Ra and injected subcutaneously in the inner thigh. This method is used to obtain primed lymph node cells for in vitro T cell proliferation assays (see Support Protocol 2). Additional Materials (also see Basic Protocol 1) 800 g ml mouse thyroglobulin (MTg) in PBS, pH 7.2, or nonpyrogenic saline...

Tumor Protection Using Gmcsftransduced Wholecell Vaccine B16gmcsf

It is difficult to induce reliable protection against B16 challenge by vaccination with irradiated B16, even when admixed with Corynebacterium parvum. However, robust protection can be obtained by vaccinating with B16 that is retrovirally transduced to secrete high levels of GM-CSF (Dranoff et al., 1993). Although B16.GM-CSF will still grow upon injection, vaccination with irradiated cells will induce a T cell-dependent protection against wild-type B16. It is unknown what antigens are targets...

Sizeexclusion Chromatography

Size-exclusion chromatography can be used to separate proteins in order of large to small molecules. Protein mixtures are applied to an SE column containing a chromatographic matrix of defined pore size. Proteins are eluted with an aqueous buffer, collected as individual chromatographic fractions, and analyzed separately. SE chromatography can separate proteins based on differences in molecular size or to desalt proteins (i.e., remove low-molecular-weight contaminants such as salts, amino...

Animal Models of IgA Nephropathy

IgA nephropathy (IgAN) is a form of immune complex glomerulonephritis that occurs spontaneously in humans. The disease is characterized by accumulation of noncovalent macromolecular aggregates within glomeruli, and is distinguished from other forms of immune complex glomerulonephritis by virtue of the predominance of IgA as the major class of Ig within the aggregates. Although IgAN is the most common form of glomerulonephritis throughout the world, its clinical expression is highly variable and...

Trampc2 Subcutaneous Tumor Resectionmetastasis Model

To facilitate studies more closely focused on prostate tumor metastasis, we recently introduced an immunocompetent murine model that nominally recapitulates clinical metastatic cancer relapse after primary tumor resection Kwon et al., 1999 . The establishment of this model is significant because, in general, the development of adjunctive cancer therapies has been hindered by the absence of such models. This model capitalizes on the capacity of TRAMP-C2 cells to metastasize to regional lymph...

Intrathymic Injection Of The Mouse

Mouse Intrathymic Injection

Scalpel handle 3 and scalpel blade 15 1-ml syringe with 28- to 30-G needle Wound clips or 4-0 suture material with needle Additional reagents and equipment for anesthesia unit 1.4 1. Anesthetize the mouse and immobilize in dorsal recumbency on a surgical board. 2. Swab the chest area with 70 ethanol on a gauze sponge or swab. 3. Make a 1-cm midline incision with scalpel and blade at the thoracic inlet i.e., at the junction of the lower cervical and upper thoracic regions. 4. Use thumb forceps...

Electricity And Electrophoresis

Many researchers are poorly informed concerning the electrical parameters of running a gel. It is important to note that the voltages and currents used during electrophoresis are dangerous and potentially lethal. Thus, safety should be an overriding concern. A working knowledge of electricity is an asset in determining what conditions to use and in troubleshooting the gel, if necessary. For example, an unusually high or low voltage for a given current milliampere might indicate an improperly...

Evaluating Eamg Using Electromyography

Electromyography Mouse

Electromyography EMG is an objective, quantitative measurement of muscle weakness it is used to demonstrate decremental responses to repetitive nerve stimulation. This test is used to diagnose myasthenia gravis MG in humans and experimental autoimmune MG EAMG in mice. In EMG, electric currents are delivered to the nerves at a rate of three per second, and action potentials are recorded from an electrode inserted into the respective muscle. Additional Materials also see Basic Protocol Styrofoam...

Detection Of Serum Autoantibody To Ovarian And Gastric Antigens Using Indirect Immunofluorescence

In this procedure for evaluating autoimmune disease, frozen sections of adult mouse ovary and stomach are used as tissue substrate, and fluoresceinated antibody to mouse IgG is used as secondary antibody. The primary antibody is serum from autoimmune, 3-day thymectomized mice. The most common ovarian antibodies react with cytoplasmic antigens of the mature and developing oocytes. Some antibodies react with the zona pellucida ZP , and occasional antibodies react with interstitial and theca...

Materials

Crude MAP system see Basic Protocol 1, 2, or 3, or Alternate Protocol 0.1 M NH4HCO3 NH4 2CO3 pH 8.0 in 8 M and 2 M urea 1 M acetic acid Dialysis tubing e.g., Spectra Por 6, MWCO 1000, Spectrum 1. Dissolve crude MAP system in 100 ml of 0.1 M NH4HCO3 NH4 2CO3 pH 8.0 in 8 M urea. 2. Load the peptide solution into dialysis tubing. 3. Dialyze the peptide solution sequentially against 2 liters of each of the following solutions, for 8 hr each, at room temperature 0.1 M NH4HCO3 NH4 2CO3 pH 8.0 in 8 M...

Frequency Determinations Using Singleparameter Fluorescence Histograms

Frequency determinations are used to calculate the number or frequency of cells with defined characteristics as measured using flow cytometry. For example, the percentage of total cells that react positively with a fluorochrome-labeled reagent is a frequency determination. In the following steps, frequencies are calculated from single-parameter histogram data files. The major requirement for performing this analysis is flow cytometer software capable of displaying histograms, allowing selection...

Direct Extraction Of Eicosanoids Into Organic Solvents

Extraction and purification of eicosanoids are likely to be necessary if there are contaminating substances in the sample that will interfere with the assay. Organic solvents, detergents, proteins, and high levels of salts can all interfere with obtaining good results. It is important to note that extraction procedures should be avoided, if possible, since they require substantial effort and always result in sample loss see Critical Parameters . This protocol describes an acetone chloroform...

Purification Of IgM By Dialysis And Sizeexclusion Chromatography

Many IgM antibodies will precipitate when dialyzed against distilled water. The IgM produced by this method is of sufficient purity to be used for fragmentation unit 2.10A , fluorescein isothiocyanate FITC or biotin labeling unit5.3 , or radiolabeling unit8.11 . The protocol involves centrifugation of ascites fluid or monoclonal antibody supernatant, followed by simple dialysis against water. IgM is separated from contaminating proteins such as normal mouse proteins or proteins from fetal...

Background Information

The BB rat strain was derived from a colony of outbred Wistar rats that developed spontaneous diabetes mellitus at the BioBreeding Laboratories, Ottawa, Canada, in the 1970s. Affected animals became the founders of the inbred diabetes-prone DP -BB Wor rat strain used in the majority of published studies. At the sixth generation of inbreeding, a subpopulation of nondiabetic DP-BB Wor rats was selected to start a control line. Now designated as diabetes-resistant DR -BB Wor rats, these...

Electrophoresis Onto Na45 Paper

This procedure is simpler than the first basic protocol, as it does not require the manipulation of gel slices and dialysis bags. It is particularly suitable for small DNA fragments i.e., lt 2000 bp . After preparative gel electrophoresis in a gel containing ethidium bromide, a piece of NA-45 paper is inserted into the gel ahead of the fragment of interest. Further electrophoresis results in migration of the fragment onto the paper, which then is removed from the gel. The fragment is eluted...

Basic Protocol

Dial Thickness Gauge Mice

Contact hypersensitivity is a simple in vivo assay of cell-mediated immune function. In this procedure, exposure of epidermal cells to exogenous haptens results in a delayed-type hypersensitive reaction that can be measured and quantified. The Langerhans cell is the critical antigen-presenting cell in this reaction this Ia , bone marrow-derived, epidermal cell initiates sensitization to haptens by presenting antigens to CD4-bearing T lymphocytes which, in turn, secrete lymphokines and recruit...

Critical Parameters

It is usually preferable to use SE chromatography for purification of IgM after dialysis, as very few contaminants have molecular weights as high as 900 kDa the molecular weight of IgM . Fast protein, peptide, and polynucleotide liquid chromatography FPLC can also be used with Superose 6 Prep Grade Pharmacia Biotech with a bed volume of 125 ml. Although it considerably reduces the time needed to run a column, FPLC equipment is expensive. Ion-exchange IEX chromatography unit2.8 can be...

Peripheral Blood Fsc

Spleen Fsc Ssc Gate

Expand the Experiment Document to full size by clicking the zoom box in the upper right-hand corner of the window. 5. Choose Dot Plot from the Plots menu. Click and hold the Plot Source box to open the pop-up menu and choose Acquisition. Click OK on the defaults for X and Y parameters FSC and SSC . 6. Repeat step 5, but choose FL1-H for Y Parameter. Click and drag the frame of the plot to position it away from the other dot plot. 7. Choose Histogram Plot from the Plots menu. Click and hold...

Purification By CsClEthidium Bromide Equilibrium Centrifugation

Ethidium Bromide Gradient

This procedure yields high-quality plasmid DNA free of most contaminants such as chromosomal DNA and RNA. Because binding of ethidium bromide lowers the density of DNA and because the covalently closed plasmid DNA can bind less than chromosomal DNA, plasmid DNA forms bands in a region of greater density lower in the tube than does the chromosomal DNA. Many investigators alternatively use commercially available columns for purification see Background Information , to avoid both the long...

Neonatal Thymectomy

Neonatal thymectomy is used most often to remove mature T cells without removing T cell precursors. Mice from birth to day 3 can be thymectomized for this purpose. Removal of the thymus after day 3 does not alter the influence of the thymus on the development of later T cell-dependent antibody or cellular immune responses. Saline or phosphate-buffered saline PBS appendix 2 Sterile 5 x 5-cm gauze sections Incandescent desk lamp Dissecting board wood or styrofoam Dissecting microscope Iris...

Purification Of IgM By Ammonium Sulfate Precipitation

This protocol can be used for most IgM antibodies. It is more time-consuming and tedious than dialysis against water, but should be used for IgM antibodies that will not precipitate in water. Ammonium sulfate precipitation of IgM is similar to that described for IgG in unit2.7 Basic Protocol 1 . This is followed by dialysis and size-exclusion chromatogra-phy as described above for IgM. Additional Materials also see Basic Protocol 1 Saturated ammonium sulfate SAS unit 2.7 Ammonium sulfate...

Isolation Of Bispecific Antibodies

The bsAb can be produced either as ascites or in culture supernatant. Ion-exchange chromatography using Bakerbond ABx columns under FPLC conditions is often successful in purifying the desired bsAb from parental antibodies and mispaired molecules. Chromatography conditions should be optimized by running the column with the parental hybridoma antibodies individually and as a mixture. Ascites fluid or supernatant containing bsAb unit 2.6 25 mM 2- N-morpholine ethane sulfonic acid MES Sigma , pH...

Culture Of Phoenix Cell Lines

It is extremely important that the packaging cells be maintained in a healthy monolayer for successful transfection and for obtaining the highest virus titer. The following protocols cover the culture, passaging, and freezing thawing methods optimal for these cells. Phoenix cells are not as adherent as familiar fibroblast lines, and they tend to clump and peel off if overgrown or if improperly treated with trypsin. Reapplication of drug selection every few months is recommended to maintain...

Adult Thymectomy

Suprasternal Notch Mice

Removal of the thymus in an adult mouse creates a status quo in the immune system no new T cells will be generated, allowing the analysis and experimental manipulation of existing T cells without any dilution by incoming T cells. Thymectomy of the adult mouse is similar to that of the neonate however, as the thymus undergoes considerable enlargement in the first few weeks of life, some adjustments in technique are required as described below. Protocols for young and adult thymectomy in rats are...

Basic De52 Ionexchange Chromatography With Triscl Protocol

DE52 ion-exchange IEX chromatography can be used to purify antibodies from a tissue culture supernatant, ascites fluid, and serum or ammonium sulfate precipitates derived from any of these antibody-containing fluids. The protocol may also be used as a second step following purification by size exclusion SE chromatography see Basic Protocol 1 and Alternate Protocols 1 and 2 . The major contaminant protein in all these preparations is albumin, which binds DE52 tightly under conditions of...

Complete RPMI medium supplemented

Prepare complete RPMI as in appendix 2a and add the following 1 mM sodium pyruvate 50 g ml gentamycin 10 mM HEPES 10 FBS To prepare Dulbecco's phosphate buffered saline DPBS , first slowly add 100 ml of 1 mg ml CaCl22 H2O to 300 ml of water while mixing. To this solution, slowly add 100 ml of 1 mg ml MgCl26 H2O and mix well. To the resultant solution, slowly add 500 ml modified PBS see recipe and mix well. Store up to 3 months at 4 C. It is important that the solutions are added in the order...

Isolation Of Monocytes By Counterflow Centrifugal Elutriation

Elutriation Centrifugation

Counterflow centrifugal elutriation CCE is a highly effective method for separating large numbers of monocytes from Ficoll-Hypaque purified PBMC unit 7.1 . The protocol has four parts assembly of the CCE system, preparation of mononuclear cells for CCE, loading of mononuclear cells, and isolation of monocytes by CCE. In contrast to the adherence and sedimentation methods of isolation, this procedure does not lead to monocyte activation. Phosphate-buffered saline PBS appendix2 low pyrogen and...

Basic Blood Collection From Tail Vein Of Mouse And Rat Using Protocol 2 Microhematocrit Tube

Lateral Tail Vein

Heat lamp or beaker containing warm water 25- to 30-G needle Microhematocrit tube Gauze sponge Additional reagents and equipment for handling and restraint unit 1.3 2. Warm the tail with a heat lamp or immerse in warm water to dilate the vessels. 3. Visualize a sampling site of the lateral tail vein at approximately midpoint on the length of the tail. 4. Extend the tail with one hand, and with the other hand insert needle 3 to 4 mm into the lateral tail vein as shown in Figure 1.7.2. 5. Collect...

Blood Collection From Orbital Sinus Or Plexus Of Mouse And

Orbital Sinus Mice

Sterile saline or phosphate-buffered saline PBS appendix 2 Microhematocrit tube Gauze sponge or swab Additional reagents and equipment for handling and restraint unit 1.3 and anesthesia unit 1.4 1. Manually restrain and anesthetize the animal. 2. Introduce the end of the microhematocrit tube at the medial canthus of the orbit as shown in Figure 1.7.1. 3. Slowly, and with axial rotation, advance the tip of the microhematocrit tube gently towards the rear of the socket until blood flows into the...

Commentary

Integral membrane proteins normally reside in a hydrophobic environment. Detergent solubilization results in a fundamental change in that environment such that these proteins can now exist in an aqueous phase. The parameters used to define a protein as being adequately solubilized vary, depending on the methodology used in subsequent procedures. In the protocols described here, the proteins are solubilized if the activity of interest remains in the supernatant after a 15-min centrifugation at...

Intravenous Injection Of The

Rat Penile Vein

1-ml syringe with 25- to 30-G needle Additional reagents and equipment for anesthesia unit 1.4 2. Fill syringe with injectate and remove air bubbles. 3. Place the rat in a supine position. Extrude the glans penis by sliding the prepuce downwards and pressing at the base of the penis. Hold the glans at the tip with the thumb and forefinger of one hand. Without applying too much tension on the penis, the penile vein is visible as a central vein or sinus Fig. 1.6.5 Waynforth, 1980 . 4. Advance...

Multipin Synthesis Of Peptides

Derivatized pins with macrocrowns or gears are attached to a pin holder. Each peptide is built up on the reactive surface of one pin by successive cycles of amino acid coupling, followed by washing and removal of the 9-fluorenylmethyloxycarbonyl Fmoc amino-protecting group to prepare for the next amino acid coupling cycle. A critical step is properly dispensing activated amino acid solutions into the appropriate wells of each reaction tray. A list of the well locations for dispensing of each...

Labeling P815 Cells For Nk Cell Adcc Assay

Rabbit anti-mouse lymphocyte Ab Accurate Chemical Additional reagents and equipment for analysis of ADCC activity unit 7.27 and counting cells by trypan blue exclusion appendix 3b 1. Incubate 1-2 x 106 P815 cells with 15 l of rabbit anti-mouse lymphocyte antibody on ice in a total volume of 100 to 300 l RPMI-10 for 15 to 20 min. 2. Wash cells with 10 ml of PBS or RPMI-1640 two times, resuspend in 200 l of RPMI-10, and count cells by trypan blue exclusion appendix 3b . It is not uncommon to have...

Nonionic Detergent Solubilization Of Lymphocytes

Detergents are organic compounds that have both hydrophilic and hydrophobic characteristics. In their monomeric forms, they are soluble in both water and lipid. Choice of a particular detergent for initial solubilization or for addition after solubilization is somewhat empirical. Educated guesses can be made, however, as to the suitability of a detergent for a particular situation based on its structure and its intended use. Commonly used detergents and their properties are described in...

Intravenous Injection Of The Rabbit

Marginal Vein Rabbit For Injection

Restrainer unit 1.3 Gauze sponge or swab 1- or 3-ml syringe with 25- to 30-G butterfly infusion set 2. Swab the ear with 70 ethanol on a gauze sponge or swab. 3. Attach the infusion set to the syringe. 4. Fill syringe with injectate and remove air bubbles. 5. Apply pressure around the base of the ear to distend the marginal ear vein. 6. Insert needle just beside and parallel to the vein. DO NOT attempt to insert the needle directly into the vein from above as this will flatten the vein, making...

Instrument Checkout Using Calibrite Beads And Facscomp Software

Standard fluorescent beads are used to check performance and alignment of the FACS Calibur. The following protocol describes the use of CaliBRITE beads and the accompanying FACSComp software both from Becton Dickinson alternatively, other brands of standard fluorescent beads can be used in conjunction with the CELLQuest software. A complete description of FACSComp is found in the FACS Calibur FACSComp Software User's Guide. FACSComp is an interactive program that performs three functions...

Construction Of Iscoms Containing Palmitified Proteins

ISCOMs are open cage-like structures 30 to 40 nm in diameter that require cholesterol and a mixture of saponins Spikoside to form. To make ISCOMs, cholesterol and Spikoside are dissolved in a solution containing detergent. On removal of the detergent by dialysis or by density-gradient centrifugation, the characteristic structure of the ISCOM forms spontaneously. An unsaturated fatty acid, such as phosphatidylcholine, must also be added to make ISCOMs with purified proteins that contain no...

Basic Footpad Injection Of The Mouse Protocol

Manually restrain the animal unit 1.3 . This procedure is usually done with one person restraining the animal while the other injects the substance. 2. Spread the toes of one hind foot and introduce the needle into the soft tissue pad of the plantar surface. Alternatively, the injection can be made subcutaneously between the second and third digits. 3. Inject no more than 50 l. Apply direct pressure following withdrawal of needle.

Induction of Immune Responses Introduction

The immune system is composed both functionally and anatomically of cellular and humoral compartments. The cellular component of immunity relies most heavily on lymphocytes dependent on the thymus for their development and maturation T cells , while the humoral component consists of a class of soluble molecules found in the serum antibodies that are characterized by enormous diversity as well as specificity. These antibodies are synthesized by non-thymic cells derived from bone marrow...