CAS No. : 9014-00-0
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|Cat. No. :||CS-7681|
|CAS No. :||9014-00-0|
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Luciferase is a class of enzyme which could oxidize D-luciferin to chemically generate visible light, and is widely used for biological assays and imaging. In Vitro: The increase in IFNγ, TNFα, and IL-2 expression observed by intracellular staining of splenocytes after exposure to Luciferase peptide plus the specific binding of MHC I-luciferase peptide tetramer to activated liver lymphocytes provides definitive evidence for a cytotoxic T cell response against Luciferase in animals infused with pT2/CaL plus SB100X, resulting in the rapid loss of Luciferase expression from around 1010 p/sec/cm2 to less than 107 p/sec/cm2. In Vivo: SCID mice are subsequently used to evaluate the Luciferase expression by in vivo bioluminescence imaging. All mice exhibite high levels of Luciferase expression (>1.5×108 and up to 3.8×1010 p/sec/cm2) 1 day postinfusion, and as previously observed there is a rapid loss of Luciferase expression in animals. All animals co-infused with pCMV-SB100X maintain high levels of Luciferase expression (>109 p/sec/cm2), without diminution from day 1 levels, which lasts for at least 4 weeks.
Protocol:Cell Assay: Following CO2 asphyxiation, animals are perfused with 0.9% sodium chloride and then the liver and spleen are harvested and disrupted into single-cell suspensions. Liver lymphocytes are isolated on a 44% to 67% percoll gradient. Red blood cells in splenocyte suspensions are lysed using ACK lysis buffer. Cell suspensions are stimulated with 1 μg/mL Luciferase peptide plus 1 μg/mL GolgiPlug for 4 hr at 37°C. Animal Administration: Mice are anesthetized with 0.2 mL of ketamine cocktail i.p., and then given 0.1 mL of 28.5 mg/mL D-luciferin i.p. Animals are then imaged for a duration of 0.5 sec to 5 min using the imaging system. Luciferase activity levels are expressed in photons emitted per second per square centimeter, denoted as p/sec/cm2.
David M. Mofford, et al. Aminoluciferins Extend Firefly Luciferase Bioluminescence into the Near-Infrared and Can Be Preferred Substrates over d-Luciferin. J Am Chem Soc. 2014 Sep 24; 136(38): 13277-13282.
Kelly M, et al. Cellular Immune Response Against Firefly Luciferase After Sleeping Beauty–Mediated Gene Transfer In Vivo. Hum Gene Ther. 2014 Nov 1; 25(11): 955-965.
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