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Detection of active apoptotic molecules with femtogram sensitivit | 20937
Journal of Cell Science & Therapy

Journal of Cell Science & Therapy
Open Access

ISSN: 2157-7013

+44 1300 500008

Detection of active apoptotic molecules with femtogram sensitivity in single cells: A pilot study


2nd World Congress on Cell Science & Stem Cell Research

November 12-14, 2012 Hilton San Antonio Airport, USA

Petra Cela, Jitka Hegrova, Marcela Liskova, Marcela Buchtova, Eva Matalova and Karel Kleparnik

Posters: J Cell Sci Ther

Abstract :

W ith an increasing interest in mechanisms of cell death also with regards to therapeutical applications, the research techniques become precised. Particularly at the protein level, where amplification possibilities are limited, novel sensitive approaches are welcome. Earlier, we demonstrated a novel combination of the laser capture microdissection and flow cytometry of cryopreserved samples (Matalova et al. 2010). Later, a modified chemiluminiscence technology with photon counting detection allowed for detection of active caspase-3 in the femtogram amount (Chlastakova et al. 2012). Now, a challenging project has been started to detect active caspase-3 in one single cell with precision of femtograms. To achieve the aims, an experimental design of embryonic and stem cells treated by camptothecin to induce apoptosis was used as described in Chlastakova et al. 2012. The developed device for the detection of caspase 3 activity in individual apoptotic cells takes advantage of Luciferin/Luciferase chemiluminescence reaction. The luciferin modified with a tetrapeptide sequence (DEVD), specific to the recognition of caspase-3, is cleaved to form free luciferin immediately reacting with luciferase to produce light. Our detection device consists of a light-proof microfluidic chamber held inside a housing of photomultiplier tube (Hamamatsu R446) with spectral response 185-870 nm. The limit of detection reaches the value lower than 1pg of caspase 3. Thus, the sensitivity of the device proved to be by one order of magnitude better than the commercially available technologies. The results show that about 1.6�10-19 mol (96,000 molecules) of caspase 3 are activated in a single apoptotic cell. The research at the IAPG CAS, v.v.i. was supported by the Grant Agency of the Czech Republic (P503/11/2315) and RVO: 67985904. The work at the IACH CAS, v.v.i. was supported by the Grant Agency of the Czech Republic (GA203/08/1680, P206/11/2377, P301/11/2055 and P206/11/P002), institute research plan AV0Z40310501 and institutional support RVO:68081715. Chlastakova I et al. 2012: In Vitro Cell Dev Biol ? An (DOI 10.1007/s11626-012-9542-8) Matalova E et al. 2012: Arch Oral Biol 55: 570?575

Biography :

Petra Cela graduated in Animal Physiology at Faculty of Science, Masaryk University in 2011. Now, she continues Ph.D. in Laboratory of Animal Embryology, Institute of Animal Physiology and Genetics, Academy of Sciences of the Czech Republic, v.v.i

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