Recombinant Fluc protein (Km = 2.06 μM )

The signal in LLC/luc and MDA-MB-231/luc cells treated with AkaLumine HCl peaked at a lower concentration (2.5 μM), while D-luciferin and CycLuc1 produced more bioluminescence at higher doses. rise, even at 250 μM not reaching the maximum signal [1].

AkaLumine hydrochloride revealed a considerably elevated signal from lung metastases, with an 8.1-fold augmentation compared to administration of D-luciferin. To evaluate the superiority of AkaLumine HCl over CycLuc1 in detecting deep tissue targets, bioluminescence of AkaLumine HCl and CycLuc1-treated mice 15 min after intravenous injection of LLC/luc cells at a 5 mM dose (maximum dose) Images for comparison The concentration of CycLuc1 is higher due to its low water solubility. Compared with CycLuc1, AkaLumine hydrochloride shows a 3.3-fold boost in detection sensitivity for disseminated lung cancer cells. The superiority of AkaLumine hydrochloride was further validated by imaging the same mice with lung metastases following intraperitoneal injection of 5 mM substrate in the order of CycLuc1 and AkaLumine hydrochloride, and every 8 hours in the reverse order. Imaging. AkaLumine hydrochloride demonstrates an approximately fourfold increase in lung metastatic signal compared to CycLuc1 [1].

Measurement of bioluminescence emission spectra [1]
Bioluminescence emission spectra of d-luciferin, CycLuc1 and AkaLumine-HCl were measured using an ATTO AB-1850 spectrophotometer. A reaction mixture was prepared by mixing 5 μl of a substrate (100 μM), 5 μl of QuantiLum Recombinant Luciferase solution (1 mg ml−1) and 5 μl of potassium phosphate buffer (500 mM, pH 8.0). Luminescence reactions were then initiated by injecting 10 μl of ATP-Mg (200 μM) into the reaction mixture. Bioluminescence emission spectra were measured in 1 nm increments from 400 to 780 nm using 3 min of integration time.[1]

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Measurement of K m value[1]
The bioluminescence intensities of d-luciferin, CycLuc1 and AkaLumine-HCl were measured in same conditions with measurement of bioluminescence spectrum, except substrate concentration. Final concentrations of the substrates were varied from 0.2 to 500 μM and 0.1 to 100 μM, respectively. Km and Vmax values of the substrates were determined from the integrated value of the bioluminescence intensity and calculated by the Lineweaver–Burk plots by using the Enzyme Kinetics Wizard in the commercially available SigmaPlot 9.0 software package.

Bioluminescence transmission assay using biological tissues[1]
Bioluminescence signal from wells was measured with IVIS Spectrum 1 min after the substrate (final concentration of 2.5 μM for d-lucfierin and AkaLumine-HCl, 50 nM for CycLuc1) was reacted with recombinant Fluc proteins (20 μg ml−1) in the presence of ATP-Mg (final concentration of 20 μM) in a black 96-well plate. A biological tissue (4-mm-thick sliced beef) was placed on the wells, to measure bioluminescence signal through the tissue, followed by acquiring bioluminescence images through two layers of the biological tissue. The following conditions were used for image acquisition: exposure time=10 s, binning=medium: 8, field of view=12.9 × 12.9 cm, and f/stop=1. The bioluminescence images were analysed by Living Image 4.3 software (PerkinElmer) specialized for IVIS system. Penetration efficiency (%) was calculated by dividing the signal intensities of the beef-covered wells by those of the corresponding uncovered wells.[1]

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Isolation of cancer cell lines stably expressing luc reporters[1]
The murine lung carcinoma cell LLC, human breast cancer cell MDA-MB-231 and human prostate cancer cell PC-3 were obtained from ATCC. LLC/luc and MDA-MB-231/luc cells were isolated after transfection with plasmid pEF/luc by calcium phosphate method as previously described19,20. PC-3/κB-luc was also isolated as previously described21. The cells were maintained at 37 °C in 5% FCS-DMEM (Nacalai Tesque, Kyoto, Japan) supplemented with penicillin (100 units per ml) and streptomycin (100 μg ml−1) and regularly checked for mycoplasma contamination by a mycoplasma check kit. All the cell lines were independently stored and recovered from the original stock every time for each experiment.[1]

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In vitro BLI[1]
The substrates were reacted with LLC/luc or MDA-MB-231/luc cells (4 × 105 cells per well) in a black 96-well plate. Bioluminescence was measured using IVIS Spectrum 1 min after adding the substrates. The following conditions were used for image acquisition: open for total bioluminescence or 680±10 nm of an emission filter for NIR bioluminescence, exposure time=10 s, binning=medium: 8, field of view=12.9 × 12.9 cm and f/stop=1. The bioluminescence images were analysed by Living Image 4.3 software specialized for IVIS system.

Tumour models
For subcutaneous tumour models, LLC/luc (3 × 105 cells per 10 μl) or PC-3/κB-luc (1 × 106 cells per 10 μl) suspended in PBS was mixed with an equal volume of Geltrex and subcutaneously or intratibially injected into C57B/6 albino mice (female) or severe combined immunodeficient mice (male), respectively. The experiments were performed 4 days after engraftment. For a model with disseminated cancer cells in the lung, C57B/6 albino mice (male) were intravenously injected with LLC/luc (1 × 105 cells per 100 μl PBS) 15 min before in vivo BLI. For lung metastasis model, LLC/luc (5 × 105 cells per 100 μl) suspended in PBS was injected from tail vein of C57B/6 albino mice (male). The experiments were performed 10–20 days after intravenous injection. These tumour models are well established and tumour growth is stable. Therefore, six samples are adequate sample size for evaluation of tumour growth in each experiment.

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In vivo BLI
Bioluminescence images of subcutaneous tumours were acquired with IVIS Spectrum 15 min (unless otherwise indicated) after intraperitoneal injection with indicated amount of the substrates. As bioluminescence intensities from lung metastasis peaked at various time after a substrate injection, bioluminescence images of lung metastasis were sequentially acquired with IVIS Spectrum every 3 min for 30 min after intraperitoneal injection with the substrates and the highest bioluminescence intensities among the acquired images were selected for analysis. For comparing bioluminescence production between different substrates using the same mice, the images for AkaLumine-HCl were acquired 4 and 8 h after injection of d-luciferin and CycLucl, respectively. The following conditions were used for image acquisition: open emission filter, exposure time=60 s, binning=medium: 8, field of view=12.9 × 12.9 cm and f/stop=1. For three-dimensional BLI in lung metastasis model, a mouse injected with AkaLumine-HCl was subjected to BLI with three different wavelengths (660±10, 680±10 and 700±10 nm) of bioluminescence filters. The following conditions were used for imaging acquisition: exposure time=60 s, binning=medium: 8, field of view=12.9 × 12.9 cm and f/stop=1. The bioluminescence images were analysed by Living Image 4.3 software specialized for IVIS system.

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Ex vivo BLI
A mouse was scarified immediately after in vivo BLI by using AkaLumine-HCl and the lung was removed. Bioluminescence image of the lung was obtained with the following conditions: open emission filter, exposure time=30 s, binning=medium: 8, field of view=12.9 × 12.9 cm and f/stop=1. The bioluminescence images were analysed by Living Image 4.3 software specialized for IVIS system.

[1]. Kuchimaru T, et al. A luciferin analogue generating near-infrared bioluminescence achieves highly sensitive deep-tissue imaging. Nat Commun. 2016 Jun 14;7:11856.

C16H19CLN2O2S

338.85

338.085576

2558205-28-8

1176235-08-7 (free acid)

Dark purple to black solid

78.2Ų

C(/C1SC[C@H](C(=O)O)N=1)=C\C=C\C1C=CC(N(C)C)=CC=1.Cl

PZCNKVAGZCXXHX-SSRSOBHISA-N

InChI=1S/C16H18N2O2S.ClH/c1-18(2)13-9-7-12(8-10-13)5-3-4-6-15-17-14(11-21-15)16(19)20;/h3-10,14H,11H2,1-2H3,(H,19,20);1H/b5-3+,6-4+;/t14-;/m1./s1

(4S)-2-[(1E,3E)-4-[4-(dimethylamino)phenyl]buta-1,3-dienyl]-4,5-dihydro-1,3-thiazole-4-carboxylic acid;hydrochloride

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: (1). This product requires protection from light (avoid light exposure) during transportation and storage.  (2). Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~125 mg/mL (~368.89 mM)
H2O : ~50 mg/mL (~147.56 mM)

Solubility in Formulation 1: ≥ 4 mg/mL (11.80 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 40.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 4 mg/mL (11.80 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 40.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)