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...

Info

Histology may vary in the different segments of the GI tract, depending on the immune status of mice. The extent of mucosal invasion can be graded (Cantorna and Balish, 1990). In the case of acute disseminated infection with an inoculum of the LD50 dose, death occurs within 4 to 7 days in BALB c and DBA 2 mice. However, parameters of infection, such as cfu recovery, mortality, and histopathology, may vary within and between mouse strains and gender, females being innately more resistant than...

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...

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...

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...

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...

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...

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...

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...

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...

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...

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...