Bioluminescence imaging (BLI) and fluorescence reflectance imaging (FRI) were performed using a NightOWL II LB 983 system equipped with a NC 100 CCD deep-cooled camera (Berthold Technologies, Bad Wildbad, Germany). For BLI, mice were injected intra-peritoneally with D-luciferin (2.9 mg in 100 μL sterile PBS, Promega), sedated at 5 min post injection and imaged at 8 min. Bioluminescence images (2 min integration period, 4 × 4 binning) and photo (100 ms exposition) were taken in prone and supine positions, respectively. A low light emitting standard (Glowell, LUX biotechnology, UK) was placed next to the animal during each image acquisition as a quality control. For FRI, images (1 s exposition, 1 × 1 binning) and photographs (100 ms exposition) were acquired. Excitation was performed at 590/20 nm and fluorescence emission was detected at 680/30 nm. Quantification was done by placing a ROI manually on the tumor and measuring the mean light intensity (in photons.s−1.cm−2) within the ROI using Indigo software (Berthold Technologies). Pseudo-color images were generated using ImageJ software.
Indigo software
Manufactured by Berthold Technologies
Sourced in Germany, United States
IndiGo is a software solution for data analysis and visualization. It provides tools for processing and interpreting data from various sources. The core function of IndiGo is to facilitate the analysis and presentation of scientific and technical data.
Automatically generated - may contain errors
Lab products found in correlation
Nightowl 2 lb983, by Berthold Technologies (6 mentions)
D luciferin potassium salt, by Promega (4 mentions)
D luciferin, by Promega (3 mentions)
Nightshade lb 985 plant imaging system, by Berthold Technologies (3 mentions)
Nightowlcam, by Berthold Technologies (3 mentions)
Nightowl lb 981, by Berthold Technologies (3 mentions)
Nightshade lb 985, by Berthold Technologies (3 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Propylene glycol, by Merck Group (2 mentions)
D luciferin potassium solution, by Berthold Technologies (2 mentions)
Balb c nu nu male mice, by Sankyo Labo Service (2 mentions)
Night owl in vivo imaging system, by Berthold Technologies (2 mentions)
Nightowl lb 983, by Berthold Technologies (2 mentions)
Luciferin, by Promega (2 mentions)
Nightowl 2 lb 983 system, by Berthold Technologies (1 mentions)
Nc 100 ccd deep cooled camera, by Berthold Technologies (1 mentions)
Originpro 8, by OriginLab (1 mentions)
Spss statistics software 20, by IBM (1 mentions)
Nightowl lb981 system, by Berthold Technologies (1 mentions)
Chitinase, by Merck Group (1 mentions)
39 protocols using indigo software
1
Bioluminescence and Fluorescence Imaging of Tumors
2
Evaluating CAR-T Efficacy in Lymphoma and Solid Tumor Models
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We established intraperitoneally injected Raji-luc lymphoma and subcutaneously implanted A549–19luc solid tumor models, in which female NOD-Prkdcscid-Il2rgnull mice (NPG/Vst, VITALSTAR) aged 4 to 6 weeks were used. The volume of cells per injection was 100 μl. CAR-T cells were resuspended in PBS and injected through the tail vein. Tumor burdens were quantified by bioluminescence imaging (BLI) on NightOwl II (LB 983, Berthold) platform, BLI data was analyzed using indiGO software (Berthold); BLI signal was reported as average flux (photons per second∕area [mm2]). All animals were anesthetized with isoflurane gas.
For tracing the T cells in peripheral blood, approximately 200 μl blood was taken through the canthus for subsequent flow analysis. To analyze the copy numbers of intratumoral CAR-T cells, the tumor masses were obtained by excision and fixed in formalin for subsequent qRT-PCR experiments.
For tracing the T cells in peripheral blood, approximately 200 μl blood was taken through the canthus for subsequent flow analysis. To analyze the copy numbers of intratumoral CAR-T cells, the tumor masses were obtained by excision and fixed in formalin for subsequent qRT-PCR experiments.
3
Luminescence Imaging of Pea Seeds
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Pea seeds were imaged by employing the NightShade LB 985 Plant Imaging System (Berthold Technologies, Bad Wildbad, Germany) equipped with a highly sensitive CCD-sensor (NightOWLcam, Berthold Technologies) thermoelectrically-cooled to −74 °C to minimise thermal noise. The camera was mounted on a dark, light-tight chamber into which the seeds to be imaged were placed (approximately 20 cm away from the camera lens). Luminescence emissions from the control seeds and weevil-treated seeds were imaged and photons emanating from the samples were detected with a back-lit, midband-coated full frame chip possessing a spectral range of 350–1050 nm (with quantum efficiency of 90% at 620 nm wavelength). During image acquisition the binning factor was set to: 2 × 2 via the software, hence, the images were captured at the resolution of 512 × 512 pixels at a final 26 × 26 µm² pixel size (slow scan mode). The exposure time was set to 60 s. For image analysis the IndiGo software (V. 2.0.5.0, Berthold Technologies, Germany) was used. Before each measurement, all the samples were kept in the light-tight dark chamber for 1 h.
The CPS values of healthy and damaged samples caused by B. pisorum were examined statistically by one-way ANOVA. Means were separated by using the Tukey (HSD) test, at p ≤ 0.05.
The CPS values of healthy and damaged samples caused by B. pisorum were examined statistically by one-way ANOVA. Means were separated by using the Tukey (HSD) test, at p ≤ 0.05.
4
Orthotopic Mouse Renal Tumor Model
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All experiments were approved by the Animal Ethics Committee of the University of Tokyo. The housing and handling conditions of the mice were consistent with the method above. Mouse renal orthotopic tumor models were conducted as previously described [22 (link), 29 (link)]. Briefly, BALB/c‐nu/nu male mice (5 weeks old) were purchased from Sankyo Labo Service Corporation (Tokyo, Japan). ccRCC cells (1.0 × 105) expressing Luc2 and mCherry were inoculated into the subrenal capsule of mice. For in vivo bioluminescence imaging, D‐luciferin potassium salt (200 mg·kg−1; Promega) was diluted in PBS and injected into mice intraperitoneally. For ex vivo bioluminescence imaging, the harvested kidneys and lungs were reacted with d ‐luciferin potassium solution for 10 min, and images were captured using NightOWL LB981 (Berthold Technologies, Bad Wildbad, Germany). Quantitative analysis was conducted using the IndiGO software (Berthold Technologies). Everolimus was reconstituted in saline solution (Otsuka, Tokyo, Japan) containing 5% Tween20 and 30% propylene glycol (Sigma‐Aldrich) and administered to mice (2.5 mg·kg−1) thrice weekly.
5
Ultra-weak Photon Emission Imaging of Cucumber Leaves
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Leaves of cucumbers grown under different nitrate supply levels were subjected to UPE (ultra-weak photon emission) imaging. Intact leaves of approximately the same size were separated from the plants and placed in the NightShade LB 985 Plant Imaging System (Berthold Technologies, Bad Wildbad, Germany). Luminescence emission of photons from the plants were visualized using a thermoelectrically cooled (−70 °C) CCD camera (NightOWLcam, Berthold Technologies) mounted on a dark, light-proof chamber. A back-lit, midband-coated full-frame chip with a spectral range of 350–1050 nm (quantum efficiency: 90% at 620 nm) was employed for photon detection and XY-imaging. To increase detection sensitivity, the variable binning was set to 2 × 2 resulting in final resolutions of 512 × 512 pixels and 26 × 26 µm² pixel size (slow scan mode). The exposure time was set to 60 s and the images were analyzed with the IndiGo software (Version 2.0.5.0, Berthold Technologies, Bad Wildbad, Germany). The presented images are a representative selection from the series of photos with the highest detected signal intensity level for each treatment. The experiment was repeated twice on different biological samples.
6
Statistical Analysis of Imaging Data
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Data analyses were performed with one-way analysis of variance using IBM SPSS statistics software 20.0 (IBM Corp., Armonk, NY, USA). Significant differences between groups were determined using Duncan’s test. The level of significance was set at 5% for all analyses. Image processing software were IndiGo software (Berthold Technologies, Oak Ridge, TN, USA) and Image-Pro plus 6.0.0 (Media Cybernetics, Inc.). Data were plotted using OriginPro 8 software (OriginLab Corporation, USA). All methods were carried out according to relevant institutional, national, and international guidelines and legislation.
7
Quantifying Tumor Burden via Bioluminescence
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The tumour burden was quantified using whole-body in vivo bioluminescence imaging with a NightOWL LB981 system (Berthold Technologies). Mice were intraperitoneally injected with 2.5 mg of D-luciferin potassium salt (Promega) dissolved in 0.2 mL of PBS. Images were acquired beginning 10 min after D-luciferin injection. Regions of interest (ROIs) were drawn around the whole body. The photons emitted from the ROIs was quantified in units of photons per second using the IndiGO software (Berthold Technologies).
8
Bimolecular Luciferase Complementation Assay
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The full‐length of GhWRKY41 was inserted into the pCAMBIA‐NLuc and pCAMBIA‐CLuc vectors to obtain GhWRKY41‐NLuc and GhWRKY41‐CLuc, respectively. The primers used are listed in Table S4 . All vectors were transformed into N. benthamiana plants via the A. tumefaciens strain GV3101. Equal amounts of Agrobacterium cultures containing CLuc and NLuc constructs were mixed, and then co‐infiltrated into N. benthamiana leaves. The infiltrated leaves were analysed for relative Luc activity 48–72 h after infiltration using a low‐light cooled charge‐coupled device camera (Night owl LB985, Germany). Quantitative analysis was performed using the IndiGo software (Berthold Technologies, Germany).
GhWRKY41‐NLuc, GhWRKY41‐CLuc and 35 S‐REN plasmid were co‐transfected into cotton protoplasts to detect the interaction of GhWRKY41 with itself. 1 mL culture solution of transformed protoplasts added 20 μL ddH2O, 20 μL V. dahliae supernatant, 20 μL 10 mg/mL chitin (sigma), 20 μL chitin+0.5 mU chitinase (sigma), respectively. The protoplasts were cultured at 25°C in the dark for 20 h and collected to measure LUC and REN value as described previously (Xiao et al., 2021a (link)).
GhWRKY41‐NLuc, GhWRKY41‐CLuc and 35 S‐REN plasmid were co‐transfected into cotton protoplasts to detect the interaction of GhWRKY41 with itself. 1 mL culture solution of transformed protoplasts added 20 μL ddH2O, 20 μL V. dahliae supernatant, 20 μL 10 mg/mL chitin (sigma), 20 μL chitin+0.5 mU chitinase (sigma), respectively. The protoplasts were cultured at 25°C in the dark for 20 h and collected to measure LUC and REN value as described previously (Xiao et al., 2021a (link)).
9
Bioluminescent Imaging of E. coli on Cucumber
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Cucumbers were cut transversely into 4 cm-disks using the kitchen knife inoculated with E. coli (7 log CFU/mL), then the bioluminescence imaging was performed. Next, each cucumber disk was longitudinally cut into 3 segments by sterile knives. Cucumber sections were then screened for bioluminescence in the dark imaging chamber at room temperature using the high-sensitivity CCD model LB of the in vivo plant imaging system (Night Shade LB 985, Berthold Technologies GmbH & Co.KG, Bad Wildbad, Germany) to monitor the inoculation area and transfer degree of E. coli. The imaging parameters for the sensitivity/resolution setup were 4 × 4 binning and a 300 s delay prior to an incremental exposure time of 180 s. Bioluminescent signals were quantified using indiGo software (Berthold Technologies, Oak Ridge, TN, USA), the light intensity was displayed by counts per second (cps).
10
In Vivo and Ex Vivo Bioluminescence Imaging
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For in vivo bioluminescence imaging, mice were anesthetized with avertin solution and intraperitoneally injected with 2.5 mg of d -Luciferin potassium salt (Promega, Madison, WI) dissolved in 200 μl of PBS. After 10 min, in vivo bioluminescence imaging was performed using the NightOWL LB981 (Berthold Technologies, Bad Wildbad, Germany). For ex vivo bioluminescence imaging, resected lungs were incubated with d -Luciferin solution for 5 min and imaged using NightOWL LB981. Images were captured and analyzed using IndiGO software (Berthold Technologies). Mice were randomly divided into groups without blinding for outcome assessment and sample size estimation. Number of mice used was indicated in each figure.
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