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- Title
INITIATION AND GROWTH OF THE AEDES AND DROSOPHILA CELL LINE CULTURES TO OBTAIN THE BIOMASS NECESSARY IN TRANSFECTION TECHNIQUE.
- Authors
VOCHIȚA, GABRIELA; DASCALU, MARIA-MAGDALENA; FUSU, LUCIAN; GHERGHEL, DANIELA
- Abstract
The aim of this study is obtaining of cellular mass from two insect species using the following cell lines: Schneider's Drosophila Line 2 (CRL-1963™) and Aedes albopictus (CCL-126™) purchased from ATCC (American Type Culture Collection), each of them having particularly growth condition. (1) Schneider's Drosophila Line 2 has epithelial origin, is derived from fruit fly (Drosophila melanogaster) embryos, was cultivated in Schneider medium supplemented with 10% heat-inactivated fetal bovine serum and 1% antibiotic solution (penicillin, 100 IU/ml and streptomycin, 100 µg/ml). The culture plates were placed in the incubator without CO2, at 23°C, the media being renewed every 2-3 days. After numerous subcultivations, during 1-2 month, the obtained cell mass was placed in a mixture of heat-inactivated fetal bovine serum (90%) and DMSO (10%), distributed in 1.0 ml cryotubes (1x106 cells/vial) and stored in an ultra-low temperature freezer. (2) Aedes albopictus cell line has epithelial origin from Asian tiger mosquito (Aedes albopictus) larvae. For cultivation, Eagle's Minimum Essential Medium (EMEM) containing Earle's Balanced Salt Solution (Earle's BSS), 0.1 mM non-essential amino acids, 2 mM L-glutamine, 1 mM sodium pyruvate, and 1500 mg/L sodium bicarbonate was used, supplemented with 20% heat-inactivated fetal bovine serum and 1% antibiotic solution (penicillin, 100 IU/ml and streptomycin, 100 µg/ml). The culture flasks were placed in an incubator at 28°C, in high humidity condition, 95% air and 5% CO2, the medium was renewed 1 to 2 times per week. After reaching 95% confluence the cells were detached using Trypsin-0.53mM EDTA solution using 1:3 subcultivation ratio. Both cell lines are considered suitable for transfection techniques. For this purpose, the insect cells were placed in contact with crude Wolbachia extracts from Diplolepis rosae larvae and centrifugated together at 2500 g, for 60 min, at 15°C followed by cultivation using the specific conditions of the respective cell line.
- Subjects
CELL lines; CELL culture; AEDES; PHYSIOLOGIC salines; DROSOPHILA; GLUTAMINE; AEDES albopictus; AEDES aegypti; ETHYLENEDIAMINETETRAACETIC acid
- Publication
Journal of Experimental & Molecular Biology, 2022, Vol 23, Issue 2, p65
- ISSN
2601-6974
- Publication type
Article
- DOI
10.47743/jemb-2022-23-2