Difference between mitogen-stimulated B and T cells in nonspecific binding of R-phycoerythrin-conjugated antibodies

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F68378041%3A_____%2F21%3A00551680" target="_blank" >RIV/68378041:_____/21:00551680 - isvavai.cz</a>

Nalezeny alternativní kódy

RIV/00216208:11310/21:10436029

Výsledek na webu

<a href="https://www.sciencedirect.com/science/article/abs/pii/S0022175921000582?via%3Dihub" target="_blank" >https://www.sciencedirect.com/science/article/abs/pii/S0022175921000582?via%3Dihub</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1016/j.jim.2021.113013" target="_blank" >10.1016/j.jim.2021.113013</a>

Jazyk výsledku

angličtina

Název v původním jazyce

Difference between mitogen-stimulated B and T cells in nonspecific binding of R-phycoerythrin-conjugated antibodies

Popis výsledku v původním jazyce

Nonspecific binding of conjugated antibodies represents a critical step which could significantly influence the results of immunostaining or flow cytometry. In this respect, various staining procedures and distinct cell types can alter the results obtained with different fluorochromes. In this study, we analysed nonspecific binding of Rphycoerythrin (R-PE)-conjugated antibodies to mouse mitogen-stimulated B and T lymphocytes. The cells were fixed, permeabilized and stained using isotype control antibodies conjugated with different fluorochromes and assessed by flow cytometry. R-PE-conjugated antibodies bound to LPS-stimulated B cells, in contrast to Con Astimulated T cells, independently of their specificity. The percentage of R-PE positive B cells varied, according to the used antibodies or the fixation/permeabilization kit. Nevertheless, up to 30% of R-PE+ B cells after staining with R-PE-conjugated isotype control antibodies was detected. Furthermore, LPS-stimulated B cells bound nonspecifically, in a dose-dependent manner, unconjugated R-PE molecules. Con A-stimulated T cells slightly bound R-PE only in high concentrations. Similarly, the antibodies conjugated with other fluorochromes showed less than 1% of nonspecific binding independently of the manufacturer of antibodies or fixation/permeabilization kits. The data demonstrated that LPS-stimulated B cells, in contrast to Con A-stimulated T cells, bind R-PE nonspecifically following formaldehyde or paraformaldehyde fixation. Therefore, the results based on the use of R-PE-conjugated antibodies should be taken with a precaution.

Název v anglickém jazyce

Difference between mitogen-stimulated B and T cells in nonspecific binding of R-phycoerythrin-conjugated antibodies

Popis výsledku anglicky

Nonspecific binding of conjugated antibodies represents a critical step which could significantly influence the results of immunostaining or flow cytometry. In this respect, various staining procedures and distinct cell types can alter the results obtained with different fluorochromes. In this study, we analysed nonspecific binding of Rphycoerythrin (R-PE)-conjugated antibodies to mouse mitogen-stimulated B and T lymphocytes. The cells were fixed, permeabilized and stained using isotype control antibodies conjugated with different fluorochromes and assessed by flow cytometry. R-PE-conjugated antibodies bound to LPS-stimulated B cells, in contrast to Con Astimulated T cells, independently of their specificity. The percentage of R-PE positive B cells varied, according to the used antibodies or the fixation/permeabilization kit. Nevertheless, up to 30% of R-PE+ B cells after staining with R-PE-conjugated isotype control antibodies was detected. Furthermore, LPS-stimulated B cells bound nonspecifically, in a dose-dependent manner, unconjugated R-PE molecules. Con A-stimulated T cells slightly bound R-PE only in high concentrations. Similarly, the antibodies conjugated with other fluorochromes showed less than 1% of nonspecific binding independently of the manufacturer of antibodies or fixation/permeabilization kits. The data demonstrated that LPS-stimulated B cells, in contrast to Con A-stimulated T cells, bind R-PE nonspecifically following formaldehyde or paraformaldehyde fixation. Therefore, the results based on the use of R-PE-conjugated antibodies should be taken with a precaution.

Druh

J_imp - Článek v periodiku v databázi Web of Science

CEP obor

—

OECD FORD obor

30102 - Immunology

Projekt

Výsledek vznikl pri realizaci vícero projektů. Více informací v záložce Projekty.

Návaznosti

I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Rok uplatnění

2021

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Název periodika

Journal of Immunological Methods

ISSN

0022-1759

e-ISSN

1872-7905

Svazek periodika

493

Číslo periodika v rámci svazku

jun.

Stát vydavatele periodika

NL - Nizozemsko

Počet stran výsledku

11

Strana od-do

113013

Kód UT WoS článku

000647677200008

EID výsledku v databázi Scopus

2-s2.0-85102367975