Explant culture of the PSM of reporter mice was performed as described previously (Masamizu et al. 2006 (link); Niwa et al. 2011 (link)). The tail regions of reporter mice were put on glass-based dishes with 1 mM luciferin (Nacalai Tesque) in 10%FBS/DMEM-F12 medium. The tissue was cultured in 80% O2 and 5% CO2 at 37°C. For image analysis, ImageJ software was used as described previously (Niwa et al. 2011 (link)). Stack images were applied to spike noise filter to remove signals from cosmic rays and then to Savitzky Golay temporal filter to get clear dynamic expression. For making spatiotemporal profiles, resliced stack images in the anterior–posterior axis were arranged from left to right in the temporal order. The spatiotemporal profiles were applied to plot profiles to obtain the dynamic reporter expression in the temporal order. Temporal difference images of Dll1 expression levels, ΔI(x,y; n), were obtained by subtracting I(x,y; n − 1) from I(x,y; n), where I(x,y; n) is a signal value at the spatial position of (x,y) and the nth frame of the temporal axis.
Luciferin
Manufactured by Nacalai Tesque
Luciferin is a bioluminescent compound that is used in various scientific applications. It is a substrate for the enzyme luciferase, which catalyzes the oxidation of luciferin to produce light. The core function of luciferin is to serve as a reagent in bioluminescence assays and experiments.
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Lab products found in correlation
5 protocols using luciferin
1
Explant Culture of Reporter Mouse PSM
2
Bioluminescent Imaging of Hes7 Oscillations
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Explant culture of the PSM of Hes7 reporter mice, pH7-UbLuc-In(−), was performed as described previously (Takashima et al., 2011 (link)). The PSM regions of reporter mice were put on glass-base dishes with 1 mM luciferin (Nacalai Tesque) in 1% BSA, 1 g/l glucose, 2 mM L-glutamine, 15 mM HEPES, penicillin, streptomycin, DMEM/F12 (Cell Culture Technologies) media and cultured in 80% O2 and 5% CO2 at 37°C. For image analysis, ImageJ software was used, as described previously (Niwa et al., 2011 (link)). Stack images were applied to a Spike-Noise Filter to remove signals from cosmic rays, and then Temporal Background Reduction was applied. Spatiotemporal profiles were obtained, as described above.
3
Bioluminescence Imaging of Neural Progenitors
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Dissociation culture of neural progenitors and bioluminescence imaging were performed as described42 (link). In brief, plasmid DNA was introduced into cultured NPCs by nucleofection with the use of an AAD-1001 device (Amaxa). The cells were plated on 35-mm glass-bottom dishes and incubated at 37°C under 5% CO2. 1 mM luciferin (Nacalai Tesque, 0149385) was then added to the culture medium. Bioluminescence images were acquired with an upright microscope (IX83, Olympus) and a cooled CCD camera (iKon-M DU934P-BV, Andor). The filters and camera control were adjusted automatically with software (MetaMorph, Universal Imaging). Image analysis was performed using ImageJ software and custom plug-ins, as described previously42 (link),86 (link). Custom plugins is available at [https://github.com/aisomur/genes_dev_2017 ].
4
Neural Progenitor Cell Bioluminescence
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Neural progenitors at E10.5–E14.5 were dissociated as described previously (Shimojo et al. 2008 (link)). They were plated into glass-based dishes with 1 mM luciferin (Nacalai Tesque) in N2/B27 medium (DMEM/F12 supplemented with 1× N2 [Gibco], 1× B27 [Gibco], 1 mM N-acetyl-cysteine, 10 ng/mL bFGF [Invitrogen]). Bioluminescence measurement was performed as previously described (Shimojo et al. 2008 (link)).
5
Quantifying Liposomal ATP Encapsulation
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Liposomes and proteoliposomes were loaded with 1 mM ATP and sonicated for 30 s using a Bioruptor (CosmoBio). After removal of the extraliposomal ATP by chromatography on Sephadex G-50 fine resin (GE Healthcare), 50-μl portions of the samples were combined with 50 μl of a mixture containing 45 nM luciferase (QuantiLum Photinus pyralis recombinant luciferase; Promega, Madison, WI), 1.2 nM luciferin (Nacalai Tesque), 1.0 mM EDTA, and 10 mM MgSO4. After 1-hour incubation with or without 0.4% Triton X-100, the luminescence was measured on an ARVO-X3 microplate reader (PerkinElmer). The amount of ATP retained in the liposomes was determined as the difference in the luminescence between total ATP and extraliposomal ATP that was accessible to luciferase without Triton X-100.
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