Abstract：
:The Rho family GTPase Cdc42 is a critical regulator of cellular polarization from yeast to man. An analysis of its function in T cell activation is therefore of interest. This analysis poses two substantial challenges, similar to the analysis of many other critical T cell signaling intermediates. First, Cdc42 is required for development and cell survival, necessitating short-term manipulation of its activity. Second, Cdc42 is likely involved in multiple signaling pathways, requiring approaches to distinguish multiple roles. To address these challenges, we first determined and quantified spatio-temporal patterns of Cdc42 activity using live cell video fluorescence microscopy. This generates hypotheses at which times and locations Cdc42 might play possibly distinct roles. Second and as the focus of this manuscript, we employed protein transduction to manipulate Cdc42 activity for the generation of causality. Protein transduction allows such manipulation to be short-term, quantitative, and with multiple reagents. Here, we characterize uptake, retention, and subcellular distribution of protein transduction reagents. We describe how a more quantitative single cell analysis of Cdc42 activity provides superior distinction between experimental conditions. And we show how we have used dose responses of the protein transduction reagents to minimize side effects while retaining efficacy. We suggest that our strategy is an important complement to more established techniques to study protein function in primary T cells, in particular in the investigation of signaling intermediates that are essential for cell survival and regulate multiple aspects of T cell activation.
journal_name
J Immunol Methodsjournal_title
Journal of immunological methodsauthors
Tskvitaria-Fuller I,Mistry N,Sun S,Wülfing Cdoi
10.1016/j.jim.2006.10.017subject
Has Abstractpub_date
2007-01-30 00:00:00pages
64-78issue
1-2eissn
0022-1759issn
1872-7905pii
S0022-1759(06)00339-5journal_volume
319pub_type
杂志文章abstract：:A number of supravital fluorochromes are available to study leucocyte functions in vitro and in vivo. The fluorescein ester most widely used, fluorescein diacetate, has the disadvantage of rapid cellular efflux, whereas more recently developed fluorescent probes do not exhibit this inconvenient trait. However, their e...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(94)90384-0
更新日期：1994-06-03 00:00:00
abstract：:In transplantation techniques previously described it was impossible to separate the effects of immunosuppressive treatments on the host from the effects directed at the allograft itself. The skin allograft retransplantation technique described here allows one to assess changes in the immunogenicity of the allograft g...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(84)90005-x
更新日期：1984-09-04 00:00:00
abstract：:The identification and characterization of human mast cell (MC) functions are hindered by the shortage of MC populations suitable for investigation. Here, we present a novel technique for generating large numbers of well differentiated and functional human MCs from peripheral stem cells (=peripheral stem cell-derived ...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2014.07.003
更新日期：2014-11-01 00:00:00
abstract：:Traditionally, the identification and quantification of eosinophils in inflammatory tissues and exudates has been primarily based upon morphologic criteria and manual counting. In this study, we describe a new flow cytometry-based assay to enumerate eosinophils present in murine bronchoalveolar lavage fluid (BAL) and ...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2007.07.011
更新日期：2007-10-31 00:00:00
abstract：:Sera from five animal species were studied as complement source in the preparation of zymosan-C3 complexes used to detect human B lymphocytes. There was no significant difference in the determination of the percentage of ZC rosette forming cells due to differences in complement source. Human B lymphocytes could not di...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(76)90195-2
更新日期：1976-01-01 00:00:00
abstract：:Dot-immunobinding assay for anti-calmodulin (CaM), and immunoblot assay for CaM have become feasible by proper choice of a fixative, i.e., formaldehyde vapor. The protocol for these simple and convenient immunological procedures is presented. Dot-immunobinding assay could be accomplished in 3 h, and immunoblot assay i...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(89)90089-6
更新日期：1989-12-20 00:00:00
abstract：:Enzyme-linked immunoadsorbent assay (EIA) has widespread use for the measurement of antibody concentration. The affinity constant (Kaff) of the antibody has an effect upon the quantification by EIA. It is thus important to be able to measure Kaff by solid-phase EIA. Based upon the Law of Mass Action and using serial d...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(87)90187-6
更新日期：1987-06-26 00:00:00
abstract：:We have developed a family of monoclonal antibodies directed against the retinoblastoma gene product (p110RB). One of these monoclonal antibodies, 3C8, binds p110RB near the C-terminal end of the protein (aa886-aa905). It was characterized by immunoblotting, ELISA, fluorescence-activated flow cytometry and immunohisto...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(94)90267-4
更新日期：1994-03-10 00:00:00
abstract：:Two highly sensitive immunoassays for measuring soluble DNA are described. These methods employ a purified mouse monoclonal anti-DNA antibody in enzyme-linked immunoassays. One assay is an antigen capture method utilizing immobilized antibody. Bound DNA is subsequently detected with biotinylated monoclonal anti-DNA an...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(86)90005-0
更新日期：1986-04-17 00:00:00
abstract：:There is an emerging trend in the pharmaceutical industry to evaluate a variety of surrogate biomarkers in Phase I/II clinical studies with the intention of determining potential activity of drugs early in clinical development. A number of cytokines expressed in pathological conditions are currently being considered a...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/s0022-1759(01)00543-9
更新日期：2002-02-01 00:00:00
abstract：:The characterization of mucins is critically important for gaining insights into the molecular pathology of diseases, including cancers, as well as for the discovery of biomarkers for disease diagnosis. However, no practical method has yet been reported for identifying mucin proteins. Here, we report a technique for i...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2013.06.002
更新日期：2013-08-30 00:00:00
abstract：BACKGROUND:ABO subgroups or weaker variants of A or B are group A or B subjects whose erythrocytes give a weak or negative reaction serologically with anti-A or Anti - B antisera respectively. Occurrence of these subgroups may lead to an ABO discrepancy which often puts transfusion services in a quandary. ABO subgroups...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2020.112895
更新日期：2020-12-01 00:00:00
abstract：:The present report describes further improvements in the methodology for antigen-specific T-lymphocyte activation in vitro. Following the experimental protocol, large numbers of enriched T-lymphocytes can be obtained from the draining lymph nodes of immunized mice after purification on a nylon column. These T-cells re...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(80)90177-5
更新日期：1980-01-01 00:00:00
abstract：:Real-time polymerase chain reaction (PCR) is now becoming an accepted tool for measuring gene expression at the transcriptional level. In this study, a direct comparison between real-time PCR, enzyme-linked immunosorbent assay (ELISA) and enzyme-linked immunosorbent spot (ELISPOT) assay was performed. When interferon-...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/s0022-1759(02)00029-7
更新日期：2002-05-01 00:00:00
abstract：:Preclinical models of lymph node (LN) metastasis are fundamental to the study and design of new techniques for the diagnosis and treatment of LN metastasis. However, the identification of LNs and lymphatic vessels (LVs) in mice is challenging with conventional imaging modalities, since the LN diameter in normal mice i...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2013.01.004
更新日期：2013-03-29 00:00:00
abstract：:The automated continuous flow system for the extraction and fluorometric analysis of histamine has been further improved. Samples with or without protein can be analyzed. The use of a single pump, added nitrogen, and lower concentration of o-phthalaldehyde increase the sensitivity by 5-10 fold and allow the analysis o...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(75)90025-3
更新日期：1975-06-01 00:00:00
abstract：:We describe an intracellular cytokine staining, flow cytometry based technique for quantitative and qualitative assessment of CD8+ T lymphocyte-mediated antigen specific immune responses. This adaptation of the technique first described by Suni et al. [J. Immunol. Methods, 212 (1998) 89; Cytometry, 40 (2000) 60] utili...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/s0022-1759(02)00025-x
更新日期：2002-04-01 00:00:00
abstract：:Human tegumentary leishmaniasis (HTL), characterized by skin ulcers that may spread and cause dreadful and massive tissue destruction of the nose and mouth, is considered a neglected tropical disease, and it is a serious threat to global health due to its continuous expansion, favored by the lifecycle of its causative...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2016.04.005
更新日期：2016-07-01 00:00:00
abstract：:Biological and biochemical characteristics of monoclonal antibodies (MABs) raised against human interleukin-10 (IL-10) are described as well as their use in the design of a specific ELISA for the measurement of the cytokine. 21 murine anti-human interleukin-10 (IL-10) MABs were obtained by fusion of splenocytes from m...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(94)90160-0
更新日期：1994-12-28 00:00:00
abstract：:Many research and clinical applications require large quantities of full-length antibodies with long circulating half-lives, and production of these complex multi-subunit proteins has in the past been restricted to eukaryotic hosts. In this report, we demonstrate that efficient secretion of heavy and light chains in a...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/s0022-1759(02)00036-4
更新日期：2002-05-01 00:00:00
abstract：:Whilst several centres have reported lymphocytotoxic antibody detection using single and dual fluorescent stains with analysis of the fluorescence emitted from the cell population present in a well of a multiwell plate, problems are encountered with cell concentration and light emission overlap. A method we have devel...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(87)90042-1
更新日期：1987-05-04 00:00:00
abstract：:Knowledge of immunodominant regions in major viral antigens is important for rational design of effective vaccines and diagnostic tests. Although there have been many reports of such work done for SARS-CoV, these were mainly focused on the immune responses of humans and mice. In this study, we aim to search for and co...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2007.11.009
更新日期：2008-02-29 00:00:00
abstract：:We describe here a spot test to detect antibodies to non-erythroid cell surface antigens, using ethidium bromide staining to visualize areas of target cell lysis. This assay allows rapid screening of large numbers of samples of culture supernatants, antisera, or ascitic fluids. Antibody titers determined by this spot ...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(82)90367-2
更新日期：1982-01-01 00:00:00
abstract：:Human properdin was prepared from the zymosan eluate or euglobulin fraction of serum by immunoadsorbent column chromatography. An immunoadsorbent column containing antibody to human serum was employed to remove serum proteins other than properdin from zymosan eluates. Immunoadsorbent with antibody to properdin was use...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(76)90095-8
更新日期：1976-01-01 00:00:00
abstract：:Near-infrared Raman spectroscopy may provide a highly sensitive, noninvasive means to identify activation status of leukocytes. The purpose of the current study was to establish Raman spectroscopic characteristics of T cell activation. Activation of the RsL.11 T cell clone in vitro with Con A resulted in specific decr...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2004.11.020
更新日期：2005-02-01 00:00:00
abstract：:Human CD1d-restricted natural killer T (NKT) cells, which are postulated to regulate the immune response in several clinical settings, can be activated by alpha-galactosylceramide (alpha-GalCer) presented by CD1d molecules on antigen presenting cells (APCs). Simple methods to quantify NKT function in fresh blood will ...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/s0022-1759(02)00497-0
更新日期：2003-01-15 00:00:00
abstract：:The indirect immunofluorescent technique has been shown to be a more sensitive method than either immunoelectrophoresis or gel filtration for testing the efficacy of the gel filtration method in fractionating human immunoglobulins. It has been confirmed that IgM fractions of some sera from patients with multiple scler...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(75)90078-2
更新日期：1975-01-01 00:00:00
abstract：:We report a method for the precise mapping of linear epitopes by presenting a peptide library on the surface of Escherichia coli cells. A random library of gene fragments derived from the classical swine fever virus (CSFV) envelope protein E(rns) was generated by DNAse I cleavage and cloned into a specially designed b...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/s0022-1759(01)00461-6
更新日期：2001-11-01 00:00:00
abstract：:The advantages of chicken (Gallus gallus domesticus) antibodies as immunodiagnostic and immunotherapeutic biomolecules has only been recently recognized. Even so, chicken antibodies remain less-well characterized than their mammalian counterparts. This review aims at providing a current overview of the structure, func...
journal_title：Journal of immunological methods
pub_type： 杂志文章,评审
doi：10.1016/j.jim.2017.05.001
更新日期：2017-08-01 00:00:00
abstract：:Immunoglobulins of the IgE and IgG classes have been causally associated with hypersensitivity reactions in man and in numerous animal species including mice, rats and guinea pigs. The use of the guinea pig as an animal model for both pulmonary and dermal hypersensitivity reactions, and the recent recognition of the i...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(91)90359-n
更新日期：1991-05-17 00:00:00
