Bioluminescence imaging was performed using the IVIS Spectrum (PerkinElmer, Villebon-sur-Yvette, France) imaging scanner coupled to the Living Image Software (Perkin Elmer) on IVIA multimodal imaging platform (Clermont-Ferrand, France) (https://doi.org/10.18145/ivia accessed on 17 May 2022). Briefly, 100 mg/kg of in vivo luciferase substrate (beetle luciferin substrate, Promega) were injected intraperitoneally into each CARE-LUC mouse. Mice were then anesthetized with isoflurane and scanned 10 min after luciferin injection. The abdominal cavity was shaved to allow for the accurate collection of bioluminescence signals. Light emission was quantified with imaging software from constant regions of interest (ROI) drawn manually. Photon emission was measured as radiance in p·s−1·cm−2·sr−1. The sensitivity of the imaging scanner was tested weekly with commercially available positive sources of bioluminescence. Excised organs were imaged immediately after euthanasia using the IVIS Spectrum (PerkinElmer) imaging scanner coupled to the Living Image Software (PerkinElmer).
Beetle luciferin substrate
Manufactured by Promega
Beetle luciferin substrate is a light-emitting compound derived from the firefly. It is used as a substrate in bioluminescence-based assays and reporter gene systems.
Automatically generated - may contain errors
4 protocols using beetle luciferin substrate
1
In Vivo Bioluminescence Imaging Protocol
2
Pseudovirus Infection Assay for HIV-1 and SIV Env-Mediated Cell Entry
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Cf2Th-CD4/CCR5 or TZM-bl cells, which express both human CD4 and CCR5, were plated at a density of 8.0 × 103 per well (Cf2Th-CD4/CCR5 cells were used in the case of 1084i Env-expressing pseudoviruses, and TZM-bl cells were used in the case of ZM249, ADA, JR-FL and SIVmac239 Env-expressing viruses). The following day, a volume of virus, normalized based on the level of RT activity in the supernatant, was added to each well in a final volume of 100 μL, and diluted in complete DMEM. One day after infection, the viral supernatants were removed, the cells were gently washed one time with PBS and fresh complete media was added to each well. On day 3 post infection, the supernatants were removed, the cells were washed once with PBS, and the cells were lysed in 1× Passive Lysis Buffer and frozen at −80 °C. The plates were then thawed and luciferase activity was measured using the Veritas Luminometer and beetle luciferin substrate (Promega).
3
Cell-Cell Fusion Assay for Env Mutants
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On Day 0, 3.5 × 105 293 T cells were plated in a 6-well plate. The following day, the cells were transfected with 2 μg of WT or mutant Env expression constructs and 1 μg Tat-expressing plasmid using polyethylynamine (PEI). Twenty-four hours following transfection, the medium was changed on the transfected cells, and TZM-bl cells were distributed in a black 96-well plate at a density of 1.0 × 104 per well. On Day 3, the transfected 293 T cells were detached from the plate using gentle pipetting and counted in a hemocytometer. Approximately 1.0 × 104 of the transfected effector cells were distributed into the wells containing the previously plated TZM-bl cells and the cells were co-incubated for 16 h. Following co-incubation, the cells were washed once with PBS, lysed using 1× Passive Lysis Buffer (Promega) and frozen at −80 °C. After thawing, he plates were read for luciferase activity using the beetle luciferin substrate (Promega) and a Veritas Luminometer. Values for the cell-cell fusion activities of WT Envs were set to 100% and mutant values are expressed as a percentage of the WT value.
4
In vivo detection of bacterial colonization
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Healthy turbot fish (average weight of ~30 g) were chosen and acclimatized in the aeration tanks for two weeks with a continuous flow of seawater at 16°C. For in vivo fluorescence detection, the subcultures of reporter strains were diluted to 106 CFU/ml in PBS. The fish were anesthetized with tricaine methanesulfonate (MS-222) (Sigma-Aldrich) at a concentration of 80 mg/l. Fish were intraperitoneally (i.p.) injected with 100 μl of bacterial suspensions. At 8 days post injection (dpi), the fish were i.p. injected with beetle luciferin substrate (Promega). After 10 min, the fluorescence was detected with a Kodak In-Vivo Multispectral System FX (Carestream Health). Then the livers of the fish were sampled and bacterial colonization was measured by CFU plating.
For competition assays, inocula were prepared using fresh cultures of bacteria that were diluted and mixed at 1:1 ratio. The injection dose was 105 CFU/fish. At 8 dpi, the livers from fish in each group (5 animals/group) were removed, homogenized and plated on DHL plates with or without containing chloramphenicol (Cm) to enumerate the ratio of the competing strains. The ratios of the bacterial counts were used to determine the CIs.
For competition assays, inocula were prepared using fresh cultures of bacteria that were diluted and mixed at 1:1 ratio. The injection dose was 105 CFU/fish. At 8 dpi, the livers from fish in each group (5 animals/group) were removed, homogenized and plated on DHL plates with or without containing chloramphenicol (Cm) to enumerate the ratio of the competing strains. The ratios of the bacterial counts were used to determine the CIs.
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