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Product Name:
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LV39 Clone 5 (+luc)
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Manufacturer:
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BEI Resources
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Taxonomy:
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Protozoa Classification: Trypanosomatidae, Leishmania
Species: Leishmania major
Subgenera: Leishmania
Strain: LV39 Clone 5 (LV39c5) (+luc)
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Additional Information:
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Leishmaniasis is caused by parasitic protozoa of the genus Leishmania, which is transmitted to both humans and animals by female phlebotomine sandflies.5,6 The sandflies inject the infective stage (promastigotes) of the parasite from their proboscis. Promastigotes that reach the puncture wound are phagocytized by macrophages and other types of mononuclear phagocytic cells. Inside the cells promastigotes transform into the tissue stage of the parasite (amastigotes) and multiply by simple division and infect other mononuclear phagocytic cells. Infection is endemic throughout the tropics, subtropics, and Mediterranean basin.5,6
The current taxonomic classification includes two subgenera, Leishmania, which are found in the midgut of the vector’s intestine, and Viannia, which are found in the hindgut of the vector’s intestine. Additionally, the more than 30 known species of Leishmania are divided into New World and Old World species, whose divergence is thought to correspond to the separation of the continents millions of years ago. The subgenera Leishmania is comprised of New and Old World species while the subgenera Viannia is comprised of only New World species.7,8 Pathogenic species of both subgenera have also been grouped into complexes based on phylogenetic analyses.9
The transfected L. major, strain LV39c5 (+luc)has been genetically engineered to express
luciferase (luc), an enzyme that emits a bioluminescent signal upon exposure to luciferin and adenosine triphosphate (ATP).2 The generation of L. major parasite lines that express bioluminescent or fluorescent proteins allows for monitoring Leishmania spp. infections using in vitro, as well as in vivo, systems.2
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Material Provided:
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Each vial of NR-50181 contains approximately 0.5 mL of culture in cryopreservative [5% dimethylsulfoxide (DMSO)]. Please refer to the Certificate of Analysis for the specific culture media used for each lot and to the Appendix for cryopreservation instructions.
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Packing/Storage:
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NR-50181 was packaged aseptically in screw-capped plastic cryovials and is provided frozen on dry ice. The product should be stored at -130°C or colder, preferably in the vapor phase of a liquid nitrogen freezer. If liquid nitrogen storage facilities are not available, frozen cryovials may be stored at -70°C or colder for approximately one week.
Note: Do not under any circumstances store vials at temperatures warmer than -70°C. Storage under these conditions will result in the death of the culture.
To ensure the highest level of viability, the culture should be initiated immediately upon receipt. Any warming of the product during shipping and transfer must be avoided, as this will adversely affect the viability of the product. For transfer between freezers and for shipping, the product may be placed on dry ice for brief periods, although use of a portable liquid nitrogen carrier is preferred. Please read the following recommendations prior to using this material.
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Growth Conditions:
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Medium 199 (M199) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (HIFBS) and 10 µg/mL hemin
Incubation:
Temperature: 25°C
Atmosphere: Aerobic
Propagation:
1. Place the frozen vial in a 35°C to 37°C water bath and thaw for approximately 2 to 3 minutes. Immerse the vial just enough to cover the frozen material. Do not agitate the vial. Do not leave the vial in the water bath after it is thawed.
2. Immediately after thawing, aseptically transfer the contents of the vial to a T-25 tissue culture flask containing 10 mL M199 medium.
3. Screw the cap on tightly and incubate the tube or flask at 25°C. Observe the culture daily under an inverted
microscope for the presence of promastigote forms of the parasite. Subculture when the culture reaches peak density.
Maintenance:
1. When the culture is at or near peak density, transfer approximately 0.1 mL to 0.2 mL into a new flask containing 5 mL to 10 mL fresh M199 medium.
2. Screw the caps on tightly and incubate at 25°C and examine daily under an inverted microscope.
3. Transfer the culture every 2 to 4 days as described in Maintenance steps 1 and 2. The transfer interval will depend on the size of the inoculum and the quality of the medium. This should be determined empirically by examining the culture on a daily basis until conditions for stable growth have been achieved. Do not allow the culture to overgrow. Viability of the culture may be affected soon after reaching peak density.
Please refer to the Appendix for cryopreservation instructions.
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Disclaimers:
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You are authorized to use this product for research use only. It is not intended for human use. Use of this product is subject to the terms and conditions of the BEI Resources Material Transfer Agreement (MTA). The MTA is available on our Web site at www.beiresources.org. While BEI Resources uses reasonable efforts to include accurate and up-to-date information on this product sheet, neither ATCC® nor the U.S. Government makes any warranties or representations as to its accuracy. Citations from scientific literature and patents are provided for informational purposes only. Neither ATCC® nor the U.S. Government warrants that such information has been confirmed to be accurate. This product is sent with the condition that you are responsible for its safe storage, handling, use and disposal. ATCC® and the U.S. Government are not liable for any damages or injuries arising from receipt and/ or use of this product. While reasonable effort is made to ensure authenticity and reliability of materials on deposit, the U.S. Government, ATCC®, their suppliers and contributors to BEI Resources are not liable for damages arising from the misidentification or misrepresentation of products. |
References:
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1. Papadopoulou, B., Personal Communication.
2. Roy, G., et al. “Episomal and Stable Expression of
the Luciferase Reporter Gene for Quantifying Leishmania spp. Infections in Macrophages and in Animal Models.” Mol.
Biochem. Parasitol. 110 (2000): 195-206.
PubMed: 11071276.
3. Al-Taqi, M., et al. “Characterization of Leishmania spp. from Kuwait by Isoenzyme
Electrophoresis.” Trans. R. Soc. Trop. Med. Hyg. 72 (1978): 56-65. PubMed: 635978.
4. Ronet, C., et al. “Leishmania major-Specific B Cells are Necessary for Th2 Cell
Development and Susceptibility to L.
major LV39 in Balb/c Mice.” J. Immunol. 180 (2008): 4825-4835. PubMed:
18354206.
5. Chappuis, F., et al. “Visceral Leishmaniasis: What
Are the Needs for Diagnosis, Treatment and Control?” Nat. Rev. Microbiol.
5 (2007): 873-882. PubMed: 17938629.
6. Reithinger, R., et al. “Cutaneous Leishmaniasis.” Lancet
Infect. Dis. 7 (2007): 581-596.
PubMed: 17714672.
7. Schönian, G., E. Cupolillo and I. Mauricio.
“Molecular Evolution and Phylogeny of Leishmania.”
Drug Resistance in Leishmania
Parasites: Consequences, Molecular Mechanisms and Possible Treatments. Eds. A. Ponte-Sucre, E. Diaz and M.
Padrón-Nieves. Vienna: Springer, 2013. 15-44.
8. Lainson, R. and J. J. Shaw. “Evolution,
Classification and Geographical Distribution.” The Leishmaniases in Biology
and Medicine. Volume I. Biology and Epidemiology. Eds. W. Peters and R.
Killick-Kendrick. London: Academic
Press, 1987. 1-120.
9. Schönian, G., et al. “Molecular Epidemiology and
Population Genetics in Leishmania.” Med.
Microbiol. Immunol. 190 (2001): 61-63.
PubMed: 11770112.
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Citation:
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Acknowledgment for publications should read "The following reagent was obtained through BEI Resources, NIAID, NIH: Leishmania major, Strain LV39 Clone 5 (+luc), NR-50181."
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Biosafety Level:
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2
Appropriate safety procedures should always be used with this material. Laboratory safety is discussed in the following publication: U.S. Department of Health and Human Services, Public Health Service, Centers for Disease Control and Prevention, and National Institutes of Health. Biosafety in Microbiological and Biomedical Laboratories (BMBL). Current Edition. Washington, DC: U.S. Government Printing Office.
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