Abstract：
BACKGROUND:High concentrations of propidium iodide (PI), in combination with fluorescein isothiocyanate (FITC) and R-phycoerythrin (RPE) used for multiparameter DNA flow cytometry (FCM), cause spectral cross-talk into the green fluorescence channel (FL1). We have evaluated the use of post-acquisition software compensation (N-Color Compensation) in order to correct this spectral cross-talk caused by PI. METHOD:Cell mixtures were prepared consisting of keratin 8/18 FITC labeled, keratin 8/18 RPE labeled, and unlabeled MCF-7 breast carcinoma cells. DNA was stained with PI (100 microM). Post-acquisition software compensation was applied to correct the spectral cross-talk of PI fluorescence. Secondly, the distribution of the Ki-67 (FITC) protein during the cell cycle (PI) of SiHa cervical carcinoma cells (no software compensation) was compared to the Ki-67 expression pattern of SiHa cells, simultaneously stained for keratin 8 (RPE), after applying software compensation. Finally, software compensation was used to compare the relative levels of PCNA and p53 expression in two clinical ovarian cancer ascites specimens, stained for PCNA or p53 (FITC), keratin 8/18 (RPE), and DNA (PI), with a known p53 status (positive and negative, respectively). RESULTS:The Ki-67 cell cycle-dependent pattern of a triply stained sample (Ki-67 (FITC), keratin 8 (RPE), and DNA (PI)) is restored after software compensation and the results are comparable to the Ki-67 distribution of a sample stained solely for Ki-67 and DNA. P53 expression could only be resolved after using software compensation in the p53 positive ovarian ascites (OA) sample. CONCLUSIONS:We conclude that software compensation is a robust and reliable post-acquisition method for the correction of RPE/PI spectral cross-talk, permitting better identification of weakly expressed proteins in heterogeneous clinical tumor samples stained for multiple cellular antigens and DNA using PI.
journal_name
J Immunol Methodsjournal_title
Journal of immunological methodsauthors
Corver WE,Fleuren GJ,Cornelisse CJdoi
10.1016/s0022-1759(01)00550-6keywords:
subject
Has Abstractpub_date
2002-02-01 00:00:00pages
97-107issue
1-2eissn
0022-1759issn
1872-7905pii
S0022175901005506journal_volume
260pub_type
杂志文章abstract：:Immune complexes attached to and ingested by human polymorphonuclear (PMN) cells were quantified by cytofluorometry using a fluorescence quenching assay which permits differentiation between attachment and ingestion. The fluorescence intensity decreased after ingestion as a result of the low pH in the phagolysosomes. ...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(83)90340-x
更新日期：1983-05-27 00:00:00
abstract：:A simplified technique for the production of chimeric thymus completely in vitro from the third pharyngeal pouch (TPP) of 10-day mouse embryos has been examined. The results are similar to those obtained by a more cumbersome technique published by others. This modified technique permits the production of large numbers...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(83)90279-x
更新日期：1983-03-11 00:00:00
abstract：:The automated complement fixation system presented in this paper differs from those described by Studievic et al. (1971) and Vargues and Henley (1974). In the one developed here only one sampler is used with a wheel presenting a double row of cups. This system can assay 60 samples per h and can be modified to double t...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(76)90183-6
更新日期：1976-01-01 00:00:00
abstract：:The neutrophilic granulocyte is the most numerous leukocyte in peripheral blood. The development from a multipotent progenitor cell to a mature neutrophil takes place in the bone marrow over a period of 10-14 days. In order to understand the cellular mechanisms behind this process, it is necessary to investigate cells...
journal_title：Journal of immunological methods
pub_type： 杂志文章,评审
doi：10.1016/s0022-1759(99)00176-3
更新日期：1999-12-17 00:00:00
abstract：:In this report the use of surface-linkage to expand the potential experimental and therapeutic applications of single chain antibody (scFv) constructs is reviewed. A strategy for the generation and functional characterization of surface-linked scFvs that bind selectively to the T-cell proteins CD3epsilon, CD28, and CD...
journal_title：Journal of immunological methods
pub_type： 杂志文章,评审
doi：10.1016/s0022-1759(00)00344-6
更新日期：2001-02-01 00:00:00
abstract：:A method that permits high energy beta-irradiation to be applied to individual lymphocyte microplate cultures is described. The principle involves the introduction of single energy sources (in this case 32P) contained within tubes which rest inside wells containing the lymphocyte cultures. Cell viability and mitogen-i...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(84)90205-9
更新日期：1984-06-08 00:00:00
abstract：:Protein A containing staphylococci were saturated with human monomeric IgG (mIgG) and cross-linked with glutaraldehyde. The resulting material (SMG) preferentially bound aggregated IgG (aIgG) and soluble immune complexes (CIC). One milliliter of a 10% suspension of SMG bound approximately 30 micrograms of mIgG and 100...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(84)90259-x
更新日期：1984-01-20 00:00:00
abstract：:In the process of metastasis, malignant cells are released from the primary tumor and migrate to specific organs via the lymphatic and blood circulation systems. These circulating tumor cells have been characterized by immunochemistry, the reverse transcription-polymerase chain reaction, and flow cytometry. Using the ...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2005.02.007
更新日期：2005-04-01 00:00:00
abstract：:Antibodies prepared by immunizing rabbits with a synthetic polypeptide representing the first 26 amino acids of the proposed NH2-terminal sequence of human erythropoietin cross-react with peptide, highly purified 125I-labeled erythropoietin and biologically active erythropoietin. We have now developed a rapid and conv...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(84)90316-8
更新日期：1984-04-27 00:00:00
abstract：:A scheme for quantitation of serum and urinary paraproteins is described using isoelectric focusing and scanning densitometry. Paraproteins could be quantified using this system when present at concentrations ranging from 1 mg/ml to 26 mg/ml, depending on the immunoglobulin class. The relevance of these results to mon...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(86)90082-7
更新日期：1986-06-24 00:00:00
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journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2014.03.014
更新日期：2014-05-01 00:00:00
abstract：:Based on the earlier observation that DNP-HSA interacts directly with C1q, a subcomponent of the first component of complement (Loos and König, 1977), evidence is presented that TNP bound to erythrocytes (E-TNP) can interact with the whole complement sequence leading to the lysis of the TNP-carrying erythrocytes; in t...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(78)90256-9
更新日期：1978-01-01 00:00:00
abstract：:The affinity of guinea pig thymocytes and peripheral T lymphocytes for rabbit erythrocytes was found to be enhanced following treatment of the erythrocytes with papain. By increasing the numbers of rosette-forming cells and giving stronger, more stable rosettes, this procedure increases the usefulness of the reaction ...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(75)90013-7
更新日期：1975-06-01 00:00:00
abstract：:An enzyme-linked immunosorbent assay was developed for quantitation of complement C3b receptors (C3bR) on human erythrocytes fixed in monolayer to microtiter plates. The disadvantages of macro test tube systems (large consumption of sample material and reagents, tedious washing procedures, cell loss and hemolysis) wer...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(85)90211-x
更新日期：1985-08-02 00:00:00
abstract：:Here we extensively describe a FACS-based protocol for isolating intact non-stained human eosinophils from peripheral blood; a stop forward from our recently published initial study. This method of purification could be accomplished in <3 h with only small volumes of whole blood necessary, even in healthy subjects gen...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2018.09.003
更新日期：2018-12-01 00:00:00
abstract：OBJECTIVES:To report on a novel immunofluorescence pattern specifically associated with antibodies to SS-A/Ro. METHODS:A novel immunofluorescence pattern, herein designated SS-A/Ro pattern, was characterized as myriad discrete fine speckles throughout the nucleus. Eighty-six sequential samples presenting the SS-A/Ro p...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2013.01.006
更新日期：2013-04-30 00:00:00
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journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/s0022-1759(02)00329-0
更新日期：2002-12-20 00:00:00
abstract：:A simple, sensitive and accurate method was developed to determine the level of lymphoid chimerism in bone marrow-transplanted rodents. The method is based on flow cytometry using polyclonal alloantisera and labeled second step anti-IgG antibodies. Using mixtures of spleen cells from different mouse strains, it was de...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(90)90044-v
更新日期：1990-07-03 00:00:00
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journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(86)90430-8
更新日期：1986-10-23 00:00:00
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journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2010.04.002
更新日期：2010-06-30 00:00:00
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journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(85)90399-0
更新日期：1985-05-10 00:00:00
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journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2006.10.017
更新日期：2007-01-30 00:00:00
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journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(90)90468-b
更新日期：1990-03-27 00:00:00
abstract：:The antigen receptor expressed on most T lymphocytes, the T cell receptor (TCR), is a multisubunit complex containing TCR alpha,beta and CD3-gamma,delta, epsilon and zeta chains, in the form alphabetadeltaepsilongammaepsilonzetazeta. The intracellular transport of TCR proteins is routinely measured by the extent of pr...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/s0022-1759(99)00103-9
更新日期：1999-10-29 00:00:00
abstract：:The sensitivity of the conventional EA (Fc) rosetting assay for detecting Fc receptors could be significantly increased by coating sheep erythrocytes (SRBC) with high concentrations of a preparation of homogeneous monoclonal IgG2b anti-SRBC antibodies. This was made possible by virtue of the fact that the monoclonal a...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(80)90218-5
更新日期：1980-01-01 00:00:00
abstract：:An assay to detect circulating immune complexes is described and exemplified with serum from SLE and RA patients. The method is based on selective precipitation of immune complexes with PEG followed by a specific ELISA assay to detect the immunoglobulins present in the precipitate. The immunoglobulins of the precipita...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(85)90186-3
更新日期：1985-02-28 00:00:00
abstract：:Cytokines are critical regulators of the development and maturation of hematopoietic cells. Signal transduction via cytokine receptors proceeds through activation of the JAK-STAT pathway to stimulate cell proliferation, differentiation and effector functions. Phosphorylation of intracellular STAT molecules by the rece...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/s0022-1759(03)00177-7
更新日期：2003-07-01 00:00:00
abstract：:We have designed a cytometry-based competition assay to evaluate peptide binding to empty recombinant HLA class II molecules. The efficiency of this assay was evaluated using recombinant HLA-DP0401 molecules (HLA-DP) produced in insect cells and 13 peptides from human telomerase reverse transcriptase (hTERT). We demon...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2011.06.018
更新日期：2011-08-31 00:00:00
abstract：:Hepatitis B surface antigen (HBsAg)-specific T cells were analysed in 16 healthy individuals vaccinated against hepatitis B, using optimised protocols. HBsAg-specific IFN-gamma producing CD4(+) T cells were found at a similar low frequency (0.01%) within the naive T cell subset, the central and the effector memory T c...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2007.10.013
更新日期：2008-01-31 00:00:00
abstract：:Cytotoxicity assays are widely used to evaluate the functional activity of NK and T cells against tumour target cells and the release of radioactive sodium chromate from labelled target cells is still the most commonly used marker of target lysis in culture supernatants. We describe here the standardization of a micro...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(94)90104-x
更新日期：1994-06-24 00:00:00
