Abstract:
:Detection of intracellular antigens by flow cytometry requires effective fixation and permeabilization of the cell membrane. This study compares three fixation/permeabilization techniques: two commercial chemical reagents, the ORTHOPermeaFix (OPF) and the FIX&PERM Cell Permeabilization Kit (F&P), and a novel method based on microwave heating (MWH). They have been applied to the detection of two nuclear (p53 and rb/p105) and two cytoplasmic (bcl-2 and mdr-1/gp-170) antigens, using positive- and negative-control cell lines and peripheral blood mononuclear cells. Western blotting was performed as a control of protein expression. For the four antigens assessed, cellular morphology, discrimination between intact cells and debris, percentage of positive cells, and mean fluorescence intensity were examined. For this last parameter, the assessment of the MWH technique was performed using SD and a graphical approach inspired by the concepts described by Bland and Altman (Lancet 1986;346: 1085-7) as well as Petersen et al. (Clin Chem 1997;43: 2039-46). The statistical analysis shows that MWH is comparable to the commercial methods and that its reproducibility is also equivalent to OPF and F&P. As assessed for some of the most clinically relevant intracytoplasmic and intranuclear antigens, the MWH method appears to be a valuable and inexpensive alternative. It is worth noting that, unlike commercial reagents, MWH altered surface antigens. Interestingly, this feature, which would prevent cell selection on the basis of combined membrane and intracellular epitopes, is associated with a decrease of nonspecific background fluorescence.
journal_name
Clin Chemjournal_title
Clinical chemistryauthors
Millard I,Degrave E,Philippe M,Gala JLsubject
Has Abstractpub_date
1998-11-01 00:00:00pages
2320-30issue
11eissn
0009-9147issn
1530-8561journal_volume
44pub_type
杂志文章abstract::A radioimmunoassay for 1,25-dihydroxycholecalciferol which did not cross react with 1,25-dihydroxyergocalciferol is described. IgG fractions were prepared from the serum of rabbits that had been immunized with 1,25-dihydroxycholecalciferol-3-hemisuccinate coupled to bovine albumin. Radioligand binding by the IgG fract...
journal_title:Clinical chemistry
pub_type: 杂志文章
doi:
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journal_title:Clinical chemistry
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journal_title:Clinical chemistry
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journal_title:Clinical chemistry
pub_type: 杂志文章
doi:
更新日期:1984-11-01 00:00:00
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journal_title:Clinical chemistry
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journal_title:Clinical chemistry
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doi:
更新日期:1979-01-01 00:00:00
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journal_title:Clinical chemistry
pub_type: 杂志文章
doi:
更新日期:1985-04-01 00:00:00
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journal_title:Clinical chemistry
pub_type: 杂志文章
doi:
更新日期:1990-01-01 00:00:00
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journal_title:Clinical chemistry
pub_type: 杂志文章
doi:
更新日期:1992-08-01 00:00:00
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journal_title:Clinical chemistry
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doi:
更新日期:1977-01-01 00:00:00
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journal_title:Clinical chemistry
pub_type: 杂志文章
doi:
更新日期:1988-08-01 00:00:00
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pub_type: 杂志文章
doi:10.1373/clinchem.2008.119396
更新日期:2009-10-01 00:00:00
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journal_title:Clinical chemistry
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doi:10.1373/clinchem.2013.214213
更新日期:2014-05-01 00:00:00
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journal_title:Clinical chemistry
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journal_title:Clinical chemistry
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journal_title:Clinical chemistry
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doi:
更新日期:1991-08-01 00:00:00
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journal_title:Clinical chemistry
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journal_title:Clinical chemistry
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更新日期:1976-11-01 00:00:00
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journal_title:Clinical chemistry
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doi:
更新日期:1993-11-01 00:00:00
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pub_type: 临床试验,杂志文章
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journal_title:Clinical chemistry
pub_type: 杂志文章
doi:
更新日期:1993-10-01 00:00:00
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journal_title:Clinical chemistry
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journal_title:Clinical chemistry
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doi:
更新日期:1976-08-01 00:00:00
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