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Statements

Subject Item
n2:RIV%2F67985904%3A_____%2F14%3A00437556%21RIV15-GA0-67985904
rdf:type
n5:Vysledek skos:Concept
dcterms:description
Caspases are key enzymes activated during the apoptotic machinery. Apoptosis as a way of programmed cell death becomes deregulated in some pathologies including cancer transformations, neurodegenerative, or autoimmune diseases. Most of the methods available for the detection of apoptosis and caspases provide qualitative information only or quantification data as an average from cell populations or cell lysates. Several reports point to the importance of more accurate single-cell analyses in biomedical studies due to heterogeneity at tissue as well as cell level. To meet these requirements, we developed a miniaturized device enabling detection and quantification of active caspase-3/7 in individual cells at a femtogram level (10(-15) g). The active caspase-3/7 detection protocol is based on the bioluminescence chemistry commercially available as a Caspase-Glo (TM) 3/7 reagent developed by Promega. As a model, we used human stem cells treated by camptothecin to induce apoptosis. Individual apoptotic cells were captured from a culture medium under a microscope and transferred by a micromanipulation system into a detection capillary containing 2 mu l of the reagent. Cells without activation by camptothecin served as negative controls. The detection limit of active caspase-3/7 achieved in the miniaturized system was determined as 0.20 and limit of quantification as 0.65 of the amount found in a single apoptotic human stem cell. Such a sensitive method could have a wide application potential in laboratory medicine and related clinically oriented research. Caspases are key enzymes activated during the apoptotic machinery. Apoptosis as a way of programmed cell death becomes deregulated in some pathologies including cancer transformations, neurodegenerative, or autoimmune diseases. Most of the methods available for the detection of apoptosis and caspases provide qualitative information only or quantification data as an average from cell populations or cell lysates. Several reports point to the importance of more accurate single-cell analyses in biomedical studies due to heterogeneity at tissue as well as cell level. To meet these requirements, we developed a miniaturized device enabling detection and quantification of active caspase-3/7 in individual cells at a femtogram level (10(-15) g). The active caspase-3/7 detection protocol is based on the bioluminescence chemistry commercially available as a Caspase-Glo (TM) 3/7 reagent developed by Promega. As a model, we used human stem cells treated by camptothecin to induce apoptosis. Individual apoptotic cells were captured from a culture medium under a microscope and transferred by a micromanipulation system into a detection capillary containing 2 mu l of the reagent. Cells without activation by camptothecin served as negative controls. The detection limit of active caspase-3/7 achieved in the miniaturized system was determined as 0.20 and limit of quantification as 0.65 of the amount found in a single apoptotic human stem cell. Such a sensitive method could have a wide application potential in laboratory medicine and related clinically oriented research.
dcterms:title
A miniaturized device for bioluminescence analysis of caspase-3/7 activity in a single apoptotic cell A miniaturized device for bioluminescence analysis of caspase-3/7 activity in a single apoptotic cell
skos:prefLabel
A miniaturized device for bioluminescence analysis of caspase-3/7 activity in a single apoptotic cell A miniaturized device for bioluminescence analysis of caspase-3/7 activity in a single apoptotic cell
skos:notation
RIV/67985904:_____/14:00437556!RIV15-GA0-67985904
n3:aktivita
n4:I n4:P
n3:aktivity
I, P(ED2.1.00/03.0101), P(GAP304/11/1418), P(GAP502/12/1285)
n3:cisloPeriodika
22
n3:dodaniDat
n13:2015
n3:domaciTvurceVysledku
n12:4002032 n12:1167405
n3:druhVysledku
n17:J
n3:duvernostUdaju
n16:S
n3:entitaPredkladatele
n15:predkladatel
n3:idSjednocenehoVysledku
829
n3:idVysledku
RIV/67985904:_____/14:00437556
n3:jazykVysledku
n10:eng
n3:klicovaSlova
apoptosis; bioluminiscence; caspase3/7; single-cell analysis
n3:klicoveSlovo
n7:bioluminiscence n7:single-cell%20analysis n7:caspase3%2F7 n7:apoptosis
n3:kodStatuVydavatele
DE - Spolková republika Německo
n3:kontrolniKodProRIV
[0013B3822864]
n3:nazevZdroje
Analytical and Bioanalytical Chemistry
n3:obor
n18:CE
n3:pocetDomacichTvurcuVysledku
2
n3:pocetTvurcuVysledku
4
n3:projekt
n8:GAP502%2F12%2F1285 n8:GAP304%2F11%2F1418 n8:ED2.1.00%2F03.0101
n3:rokUplatneniVysledku
n13:2014
n3:svazekPeriodika
406
n3:tvurceVysledku
Lišková, M. Klepárník, K. Matalová, Eva Adamová, Eva
n3:wos
000340877200017
s:issn
1618-2642
s:numberOfPages
6
n14:doi
10.1007/s00216-014-7949-7