Isolation and Functional Analysis of Neutrophils
This unit describes the isolation of neutrophils and the assays that can be performed to assess their function. Neutrophil isolation. Neutrophils or polymorphonuclear leukocytes PMN can be isolated in large numbers and with high purity from the blood of humans and many other species, as described in the first basic and alternate protocols. It should be noted that neutrophils are terminally differentiated, and thus are short-lived cells that cannot be propagated in tissue culture. Therefore,...
Crosslinking Membrane Proteins
Cross-linking membrane proteins to each other and to extracellular ligands is used to determine the physical relationship between proteins prior to solubilization and subsequent analysis see above and units 8.2-8.6 . Cross-linking can be accomplished by a number of different bifunctional reagents and conditions depending on the particular study. Initial assessment should include a trial with several cross-linkers at several concentrations. This protocol can be used before all of the above...
Info Nzb
Table 5.4.3 Troubleshooting the FACS Calibur If the Status window reads READY If the Status window reads STNDBY If the Status Window reads NOT READY Threshold parameter gain set too high or low Threshold not set to correct parameter usually FSC Sample injection tube clogged Communication failure between computer and FACS Calibur instrument GPIO error, cannot read instrument status The RUN fluid control button is not activated Sample tube cracked Sheath reservoir cap not tightened Vent valve...
Background Information Jhu
The ELISPOT assay for detecting individual cytokine-secreting cells is based on the use of highly specific monoclonal and polyclonal antibodies to human and murine cytokines. The procedure is based on the ELISPOT assay, initially described to detect individual B cells secreting antibody, for use in polystyrene plates with alkaline phosphatase Sedgwick and Holt, 1983 or horseradish peroxidase Czerkinsky et al., 1983 and later modified for use on nitrocellulose plates Moller and Borrebaeck, 1985...
Hemisplenectomy
This procedure is essentially the same as splenectomy, except that only part of the spleen is removed. 1. Follow steps 1 to 5 of the basic protocol for splenectomy. 2. Minimize bleeding by placing a small loop of suture across the center of the spleen, leaving the hilar vessels intact. Gently tighten the suture and tie it. Tightening too quickly will split the spleen, especially if there is moderate splenomegaly prior to surgery. 3. Cut the lower part of the spleen with scissors and remove it....
PURIFICATION OF MURINE IgD BY LECTIN AFFINITY CHROMATOGRAPHY
IgD is present in mouse serum at very low concentrations lt 1 g ml . Therefore, plasmacytoma and hybridoma sources are typically used to obtain milligram levels of isolated IgD. The following protocol describes a lectin affinity chromatography method for purifying myeloma-derived or monoclonal murine IgD. This method exploits the carbohydrate moieties associated with this Ig isotype and employs the a-galactopyrano-syl-binding lectin from the seeds of Griffonia simplicifolia-1 GS-I formerly...
Chemically Crosslinking Antibodies With Spdp
In this protocol two IgG antibodies or their Fab fragments are cross-linked using the heterobifunctional compound SPDP Fig. 2.13.1 . This reagent binds randomly to e-amino groups on lysine residues and forms reducible disulfide bonds between antibodies. The resulting bispecific molecules consist of aggregates of antibodies of varying size linked together at random sites. SPDP, Pierce or Sigma 100 ethanol 1 to 5 mg each of two purified IgG MAbs, affinity-purified conventional IgG antibodies...
Differential Centrifugation By Velocity
This is the classical standard method of cell fractionation using centrifugation, often used as the starting point for a more complex purification procedure. A cell homogenate is subjected to serial centrifugations of increasingly higher force and longer duration, generally in isoosmotic 0.25 to 0.3 M sucrose medium see Background Information for discussion of sucrose concentration . Resulting supernatant fractions or pellets often prepared as a band atop a cushion of high-density medium rather...
Metrizamide 145 wv
Place 25 g metrizamide Sigma 99 pure, grade 1 in a sterile 250-ml graduated cylinder. Rinse the bottle with 150 ml complete RPMI-10 appendix 2 and add this to the graduated cylinder. Dissolve the powder and then bring the total volume to 172 ml with more medium. Filter sterilize the solution into a sterile container and store at 4 C protected from light. The density of metrizamide is critical for optimal cell separation. Each newly prepared batch should be evaluated to determine that it...
Info Bqj
NKp30 shares several features with NKp46. It is selectively expressed by resting and activated NK cells and mAb-mediated crosslinking induces the activation of NK cells lytic machinery, Ca2 flux, and cytokine production Pende et al., 1999 . NKp30 is a 30-kDa glycoprotein belonging to the Ig-SF, characterized by a single extracellular Ig-like domain of the V type Fig. 14.10.1 . The transmembrane region contains an arginine residue probably involved in the association with ITAM containing CD3Z...
Info Pno
epithelial cell line, I407, grown in 25-cm2 tissue culture flasks however, it can be adapted to any in vitro system. I407 epithelial cell line ATCC-CCL6 grown in DMEM in 25-cm2 tissue culture flasks Dulbecco's minimum essential medium DMEM appendix 2 3H Arachidonic acid Amersham or NEN Life Sciences 25 g ml prostaglandin B2 PGB2 in ethanol Solvent A 10,000 1 v v HPLC-grade water acetic acid Solvent B 10,000 1 v v HPLC-grade acetonitrile acetic acid HPLC equipped with two pumps and a 4.6 x...
Alternate Protocol Tnp
A positively charged nylon membrane does not have to be prewetted, but can be placed directly onto the gel. If uncharged nylon is being used, use 0.25 M NaOH 1.5 M NaCl as the transfer solution. Check that the paper towels are resistant to the alkali solution some types go brown and should not be used. 4. Remove the paper towels and filter paper and recover the membrane. Rinse the membrane in 2x SSC, place on a sheet of Whatman 3MM filter paper, and allow to air dry. Store as described in step...
Info Ucy
Figure 3.19.4 Yield of lamina propria lymphocytes after incubation of intestinal tissue pieces in medium containing 300 U ml collagenase. The number of viable lymphocytes released into the medium was determined every 30 min. The intestines were handled in pairs. The mean values SD of seven experiments are shown. Isolation of Mouse Small Intestinal IEL, Peyer's Patch, and Lamina Propria Cells man et al., 1992 Hornquist and Lycke, 1993 Hornquist et al., 1993 . Currently it is unknown why LP T...
Preparation Of Gradient Minigels
Polyacrylamide gradients not only enhance the resolution of larger format gels but also greatly improve protein separation in the small format Matsudaira and Burgess, 1978 . Casting gradient minigels one at a time is not generally feasible due to the small volumes used, but multiple gel casters make it easy to cast several small gradient gels at one time. The gels are cast from the bottom in multiple casters, with the light solution entering first. This is the opposite of casting one gel at a...
Fluorescent Protein Stain Using Sypro Ruby For 2d Gel Analysis
Fluorescent gel stains are gaining popularity due to the combination of simplicity and sensitivity that they offer. SYPRO Ruby protein gel stain is an ultrasensitive, fluorescent stain for the specific detection of proteins separated by polyacrylamide gel electrophoresis PAGE . This stain, designed especially for use in 2-D PAGE Fig. 8.9.1 unit 8.5 , has proven to be the most sensitive protein gel stain for standard 1-D SDS-PAGE unit 8.4 and an excellent stain for isoelectric focusing IEF gels...
Classical Pathway Evaluation
This unit describes several assay methods that can be used to determine the functional status of the classical pathway of complement and to quantitate its component proteins. The classical pathway includes Clqrs, C2, C4, C3, C5, C6, C7, C8, and C9, listed in the order in which they interact. The CH50 is a simple hemolytic assay that tests total classical pathway function. Two CH50 assay procedures are presented in this unit. The first basic protocol describes a CH50 assay carried out in test...
Immunoisolation Of Endosomal Fractions
Immunoisolation can be used to purify any subcellular fraction as long as a specific antibody is available. The specific antibody is coupled to magnetic or polyacrylamide beads with a covalently coupled species-specific linker antibody, and then the beads are used to precipitate the desired organelle. Since this protocol was introduced Howell et al., 1989a , a similar strategy has been used to immunoisolate a long list of organelles and vesicles from different cell types with different...
Info Iog
4. 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...
Choice Of Transfection Method
There are two types of transfections that are routinely done in mammalian systems transient and stable or permanent. In a transient transfection, transcription or replication of the transfected gene can be analyzed between 1 and 4 days after introduction of the DNA, generally by harvesting the transfected cell. Alternatively, many experiments require formation of cell lines that contain gene s that are integrated into chromosomal DNA resulting in a stable or permanent transfection. unit 10.17A...
Infection Of Adherent Cells
The following protocol describes the infection of the murine fibroblast cell line NIH 3T3 with retroviral supernatant generated in Phoenix packaging cells according to Basic Protocol 1. The method can be applied to other adherent cell lines, and is equally efficacious with the Phoenix-amphotropic or -ecotropic packaging cell lines, or with VSV-G-pseudotyped supernatant from Alternate Protocol 1. NIH 3T3 cells should be considered a standard for infectability, although other exponentially...
Ch50 Assay For Total Classical Pathway Hemolytic Activity
The CH50 assay is the simplest quantitative functional assay for total hemolytic complement in serum or other fluid samples. It depends on sequential activation of the classical pathway components Cl through C9 to lyse sheep erythrocytes E that have been sensitized with optimal amounts of rabbit anti-sheep erythrocyte antibodies A to make cellular antigen-antibody complexes EA Mayer, 1961 Rapp and Borsos, 1970 . Complement activity is quantitated by determining the serum dilution required to...
Support Protocol 2 1
Reporter Gene Cell Fusion Assays for HIV Env Funcation 2. Optional. To ensure complete cell lysis, freeze the plate containing the samples on a bed of dry ice, then thaw at room temperature. Frozen samples can be stored up to several weeks at -20 C and thawed at a later time for -gal analysis. 3. Add 50 l of each cell lysate to individual wells of a fresh 96-well assay plate and equilibrate to room temperature. The amount of cell lysate can be varied using EMEM-2.5 as a diluent, but the total...
Info Aqa
double-ended locking hub Luer-Lok connector or 3-way stopcock Figure 2.4.1 Double-syringe device for preparation of antigen-adjuvant emulsions. 4. Transfer all of the adjuvant-antigen emulsion to one syringe and remove the connector or stopcock. Attach a 22-G needle to the syringe and remove air bubbles. 5. Restrain the animal and inject the adjuvant antigen emulsion into multiple intramuscular i.m. , intradermal i.d. , or subcutaneous s.c. sites. Discard the unused immunogen. For extremely...
CONSTRUCTION OF A scFv REPERTOIRE IN A PHAGEMID VECTOR
In this protocol, the DNA sequences of the antibody variable domains that are present in an immune mouse spleen or hybridoma cells are amplified by RT-PCR. The amplified antibody domains are then combinatorially assembled as scFv constructs and inserted into a phagemid vector. The vector is then introduced by electroporation into E. coli cells that are ready for rescue with a helper phage to produce the library of phage antibodies for affinity selection see Basic Protocol 2 and Alternate...
Measurement Of Eicosanoids By Elisa
Commercial ELISAs are commonly used to measure eicosanoids in samples. Each ELISA measures a single eicosanoid. The investigator needs to know which eicosanoids are produced by the tissue or cell type being tested, since most cell types produce more than one eicosanoid. Eicosanoid ELISAs are competition assays in which the free analyte in the sample e.g., prostaglandin E2 PGE2 and the enzyme-analyte complex e.g., PGE2-acetylcholinesterase Pradelles et al., 1985 or PGE2-horseradish peroxidase...
PURIFICATION BY CsClETHIDIUM BROMIDE EQUILIBRIUM CENTRIFUGATION
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...
Cloning By Limiting Dilution
Monoclonal antibodies are secreted by the progeny of a single cell that can produce only a single antibody assuming a nonsecretory fusion-partner line . Cloning is required to ensure that the problems of polyspecificity are avoided and the risk of overgrowth by nonproducing cells minimized. Although cloning can also be performed by the soft-agar technique, clones derived by this technique must be adapted to liquid culture before the supernatants can be tested Coffino et al., 1972 . Since...
Info Yar
gle-copy sequence that is readily visible on an ethidium bromide-stained gel. It is possible that other minor products will also be visible. The basic protocol can be completed in a single day. Assembly of the reaction mixtures should take 1 hr. Cycling should take less than 3 hr. Preparing, running, and staining the gel should take another few hours. Further checks on specificity of the product such as restriction endonuclease digestion or Southern blot hybridization will take another few...
Preparation Of Nucleoside Triphosphates
Nucleoside triphosphates have a limited shelf life in solution and hence should be stored as aliquots at -20 C, where they are stable for up to 1 year. They should be HPLC- or comparably purified. Pharmacia is the recommended supplier. Deoxyribonucleoside triphosphates dNTPs or ribonucleoside triphosphates NTPs can be purchased as ready-made 100 mM solutions, which is the preferred method of shipping and storage. Alternatively, they can be purchased in lyophilized form and prepared in deionized...
Isolation Of Monocytes By Adherence
In this protocol, monocytes are isolated from mononuclear cells by exploiting their ability to adhere to glass or plastic. This is a quick and easy procedure, but it can induce cell activation see background information . Peripheral blood mononuclear cells PBMC unit 7.1 Serum-free DMEM e.g., GIBCO BRL 320-1960 , supplemented with 2 mM L-glutamine and 50 g ml gentamycin 0.02 EDTA in PBS appendix 2 low pyrogen and without Ca or Mg 75-cm2 tissue culture flasks Falcon 3023 Beckman GPR centrifuge...
Coupling Of Peptides To Affigel 10 Affinity Matrix
This protocol describes coupling of phosphopeptides or nonphosphopeptides to Affi-Gel 10 affinity matrix for use in affinity column chromatographic purification of antibodies. Anhydrous coupling, detailed here, is the most efficient coupling method for peptides. The steps here result in production of 3 ml final bed volume of affinity resin 3 mol of peptide is coupled per milliliter Affi-Gel 10. According to the manufacturer's specifications, Affi-Gel 10 resin contains 15 mol of active ester per...
Removal Of Residual Phenol Chloroform Or Butanol By Ether Extraction
DNA solutions that have been purified by extraction with phenol and chloroform first basic protocol or concentrated with sec-butanol second support protocol can often be used without ethanol precipitation for enzymatic manipulations or in gel electrophoresis experiments if the organic solvents are removed by extraction with ether. Traces of ether are subsequently removed by evaporation. This procedure is useful only if the solute concentrations in the starting solution are compatible with what...
Culture For Antigeninduced T Cell Proliferation
This protocol is designed to test the proliferation of T cells in response to a specific antigen tetanus toxoid. It can be modified to test T cell proliferation in response to any protein or polysaccharide antigen. T cell suspension units 7.2 amp 7.4 Autologous antigen-presenting cell suspension non-T cells unit 7.2 Tetanus toxoid solution Connaught or State Laboratory Institute of Massachusetts 1. Count T cells and adjust to 1 x 106 cells ml with complete RPMI-10 AB. 2. Treat...
Safety In The Human Immunology Laboratory
Immunologic study of the human immune system poses special safety problems associated with the risk for infection with human disease agents. Attention in recent years has focused on possible infection with the AIDS HIV-1 virus. It should be noted, however, that human materials may harbor other dangerous pathogens, including hepatitis B virus HBV , cytomegalovirus, EBV, and a host of bacterial pathogens. The basic framework for safety in immunologic research is encompassed in the concept of the...
Elution In Octyl Pdglucoside
The detergent octyl P-D-glucoside has a high critical micelle concentration CMC of 0.73 and can therefore be readily removed by dialysis. Also, adsorption of membrane proteins to surfaces for ELISA or adhesion assays is much more efficient when solutions containing membrane proteins are diluted so that the concentration of octyl P-D-glucoside is below the CMC. Because octyl P-D-glucoside is expensive, initial steps requiring large volumes may be done as described in the Basic Protocol the...
Isolation of Murine Macrophages
Macrophages isolated from murine peritoneal cavity, bone marrow, and spleen are suitable samples for studying the activation properties of this immunologically important cell type. The peritoneal cavity provides an accessible site for harvest of fair numbers of resident macrophages. However, the number of macrophages present in the noninflamed peritoneum is insufficient for large-scale studies. To increase macrophage yield, sterile inflammatory agents, such as proteose peptone or...
Alternative Pathway Evaluation
The alternative pathway of complement shares its terminal components C3 and C5 through 9 with the classical pathway unit 13.1 but has several unique components, including factors D, B, and P properdin . As discussed in Background Information, it may often be desirable to assay alternative pathway function as well as or instead of classical pathway function unit 13.1 . This unit presents methods for assaying total alternative pathway activity first basic protocol and the activity of factors B...
Support Assay For Lactate Dehydrogenase To Measure Granule Protocol Release By
Although induction of granule release by stimulation of CTL specifically via anti-TCR antibodies or nonspecifically with phorbol esters and ionophores has proven to be a very useful method to measure CTL activation, one must ensure that granule release is not due to cell death induced by the activating agent. An easy method to measure cell damage-related release of intracellular components is to assay for the presence of the enzyme lactate dehydrogenase LDH . Additional Materials also see Basic...
Reagents And Solutions Nww
2 w v BSA fraction V ICN Biomedicals CAUTION Sodium azide NaN3 is poisonous wear gloves. 0.1 M Tris-Cl, pH 9.5 appendix 2 0.1 M NaCl 5 mM MgCl2 66 Ml 10 ml NBT substrate 0.4 mM Promega 33 Ml 10 ml BCIP substrate 0.4 mM Promega Prepare immediately before use 1 ml 1 M Tris-Cl, pH 8.0 0.04 mM 2.3 ml 3 M NaCl 0.276 M 10 ml 50 v v glycerol 5 ml 10 v v NP-40 25 Ml 50 mM p-nitrophenyl guanidinobenzoate 0.05 mM Sigma 50 Ml 10 mg ml aprotinin Boehringer Mannheim 50 Ml 10 mg ml leupeptin Boehringer...
Materials Yqn
Heparinized blood or heparinized cord blood appendix 3f PBS appendix 2 Ficoll-Hypaque solution density 1.077 g liter see recipe Hanks balanced salt solution HBSS appendix 2 FBS e.g., HyClone , with or without heat inactivation 1 hr, 56 C appendix 2 Complete RPMI-10 medium appendix 2 15- or 50-ml conical centrifuge tubes Beckman GPR centrifuge with GH-3.7 horizontal rotor or equivalent temperature-controlled centrifuge Additional reagents and equipment for counting cells and trypan blue...
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...
Carbonate coating buffer
1.59 g Na2CO3 0.015 M 2.93 g NaHCO3 0.035 M Add H2O to 900 ml Adjust to pH 9.6 with 1 M NaOH or 1 M HCl Add H2O to 1 liter Prepare fresh every other week because buffer absorbs CO2 and changes pH. Measurement of Mouse and Human IFN-y 0.49 g NaH2PO4 anhydrous 0.004 M 15.08 g Na2HPO4 anhydrous 0.106 M 90 g NaCl 1.5 M 5 ml Tween 20 0.5 v v Add H2O to 900 ml Adjust pH to 7.4 using either 1 M NaOH or 1 M HCl Add H2O to 1 liter Highly purified recombinant human or murine IFN-y is commercially...
Immunology By Rud
Additional reagents and equipment for counting viable cells appendix3b , labeling cells and determining 3H thymidine incorporation appendix 3b , and quantitation of interleukin activity unit 6.3 1. Prepare 96-well microliter plates with the standards and the sample dilutions as described in the Basic Protocol, steps 1 to 3. While the protocol outlined here employs complete DMEM-10 medium in conjunction with an 8 CO2 culture atmosphere, complete RPMI-10 appendix2a can alternatively be used with...
Construction Of A Calibration Curve For A Logarithmic Amplifier
This procedure describes a simple means of correlating data output from logarithmic amplifiers with precalibrated values of a fluorescent-bead standard. While higher-precision calibration procedures have been described Parks et al., 1988 Schmid et al., 1988 , the graph produced by this method is extremely valuable, and can be used to estimate the number of channels which represent, for example, a 2-fold change in fluorescence intensity. If fluorochrome-conjugated standards have been used, a...
Cell Fusion And Selection Of Hybridomas
While animals should be immunized as soon as the decision has been made to produce a monoclonal antibody and the antigen prepared, do not perform cell fusion until the screening assay first support protocol has been perfected. Artifactual results that may arise from conditioned media must be identified before cell fusion, because after a fusion there is only a finite amount of time available to assay for the desired monoclonal antibody. Prior to cell fusion, the partner myeloma cell line is...
Info Pjd
Part of the membrane was allowed to dry out during hybridization or washing Membranes adhered during hybridization or washing Walls of hybridization bag collapsed on to membrane Not enough wash solution Hybridization temperature too low with an RNA probe Labeled probe molecules are too short Probe not denatured Appropriate membrane type Unincorporated nucleotides not removed from labeled probe Particles in the hybridization buffer Agarose dried on the membrane Baking or UV crosslinking when...
Fitc
0.5 FITC in 1 1 acetone dibutyl pthalate 400 l 0.5 FITC in 1 1 acetone dibutyl pthalate 20 l 3 oxazalone in 100 ethanol 150 l 1 oxazalone in 100 ethanol 20 l 0.5 DNFB in 4 1 acetone olive oil 20 l 0.2 DNFB in 4 1 acetone olive oil 20 l Abbreviations DNFB, 2,4-dinitro-1-fluorobenzene FITC, fluorescein-isothio-cyanate Oxazalone, TNCB, 2,4,6-trinitrochlorobenzene. bOxazalone has limited solubility in ethanol and other solvents . Abbreviations DNFB, 2,4-dinitro-1-fluorobenzene FITC,...
Preparation Of Vesicular Stomatitis Virus Stocks
For this immunoisolation experiment, the antigen is provided by the cytoplasmic domain of the transmembrane glycoprotein G of the enveloped virus, vesicular stomatitis virus VSV . VSV stocks are produced and harvested as described in Gruenberg et al. 1989 . Briefly, subconfluent 2-day cultures of BHK cells are infected with 0.2 pfu VSV cell and incubated 19 to 20 hr at 37 C. Medium containing virus is removed from the cells and centrifuged 30 min at 7000 x gavg in an HS4 rotor Sorvall to remove...
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...