Journal: Cell transplantation

Article Title: Optimized Longitudinal Monitoring of Stem Cell Grafts in Mouse Brain Using a Novel Bioluminescent/Near Infrared Fluorescent Fusion Reporter.

doi: 10.1177/0963689717739718

Figure Lengend Snippet: Fig. 1. Generation of new fusion luciferase (luc2) reporters with near infrared fluorescence proteins. (A) Schematic drawing of the luc2 fusion with iRFP720 via a 14-aa linker. Photinus pyralis luc2 (PDB ID code 1LCI) and Chromophore-binding domain (CBD) of bacterial phytochrome RpBphP2 (PDB ID code 4E04) were used. (B) Western blot results using an anti-luc2 primary antibody on human embryonic kidney (HEK)-293 lysates used for transient expression of the proteins. The band in the first lane corresponds to luc2 protein (62 KDa), the second to the TurboLuc protein (*88 KDa). The third and fourth lanes correspond to luc2-iRFP670 (abbreviated pLuc670) and luc2- iRFP720 (abbreviated pLuc2720; *97 KDa). (C) Immunofluorescence for the analysis of colocalization of luc2 (green signal) and near infrared proteins (red signal) in HEK-293 cells. Nuclei are stained with 40,6-diamidino-2-phenylindole (blue signal). luc2 was detected using an anti-luc2 antibody and Fluorescein isothiocyanate (FITC)-labeled secondary antibody. Near infrared proteins were detected using a 633-nM excitation laser. Image on the left corresponds to cells expressing luc2-iRFP670 and image on the right corresponds to cells expressing luc2-iRFP720.

Article Snippet: Immunoblotting was performed after membrane blocking using rabbit polyclonal luc2 antibody (1:200; Fitzgerald Industries International, Acton, MA, USA).

Techniques: Luciferase, Fluorescence, Binding Assay, Western Blot, Expressing, Immunofluorescence, Staining, Labeling

Journal: Cell transplantation

Article Title: Optimized Longitudinal Monitoring of Stem Cell Grafts in Mouse Brain Using a Novel Bioluminescent/Near Infrared Fluorescent Fusion Reporter.

doi: 10.1177/0963689717739718

Figure Lengend Snippet: Fig. 2. In vitro and in vivo characterization of different fusion proteins. (A) In vivo fluorescence imaging of different amounts of human embryonic kidney-293 cells (blue circles) expressing TurboLuc (upper panel), luc2-iRFP670 (middle panel), and luc2-iRFP720 (lower panel). Red circle indicates autofluorescent food signals from stomach. (B) Quantification of the signal given by 1 105 cells. TurboLuc expressing cells show higher total signal. However, background signals are lower when using iRFP720 protein. Data are represented as mean (SD) (n ¼ 3).

Article Snippet: Immunoblotting was performed after membrane blocking using rabbit polyclonal luc2 antibody (1:200; Fitzgerald Industries International, Acton, MA, USA).

Techniques: In Vitro, In Vivo, Fluorescence, Imaging, Expressing

Journal: Cell transplantation

Article Title: Optimized Longitudinal Monitoring of Stem Cell Grafts in Mouse Brain Using a Novel Bioluminescent/Near Infrared Fluorescent Fusion Reporter.

doi: 10.1177/0963689717739718

Figure Lengend Snippet: Fig. 4. Histological staining and immunofluorescent staining of luc2-iRFP720 expressing human mesenchymal stem cells (hMSCs) in determining differentiation capabilities. (A) Oil Red O staining showed the presence of fat deposition, toluidine blue staining showed the presence of proteoglycans, and alizarin Red S showed the presence of calcific deposition after adipogenic, chondrogenic, and osteogenic differentiation of luc2-iRFP720 expressing hMSCs, respectively. (B; left column) 40,6-diamidino-2-phenylindole (DAPI) staining showed cell nuclei, (middle column) hMSC luc2-iRFP720 showed immunofluorescent staining of anti-mouse fatty acid binding protein 4 (anti-mFABP4), anti-human aggrecan (anti-hAggrecan), and anti-human osteocalcin (anti-hOsteocalcin) after conditioned adipogenic, chondrogenic, and osteogenic differentiation, respectively. (Right column) Merged images of DAPI and the specific antibody (n ¼ 3). (C) alkaline phosphatase (ALP) activity measured in medium of luc2-iRFP720 expressing hMSCs, and non transduced hMSCs after 14 d of adipogenic or osteogenic ALP activity during osteogenic differentiation are significantly higher (P < 0.01) than during adipogenic differentiation or undifferentiated control cells. (D) Oil Red O absorbance values at 540 nM suggest that the level of adipocytes in different culturing conditions is not affected by transduction, but it is significantly higher in adipogenic differentiation compared to osteogenic differentiation or undifferentiated cells (*P < 0.05 and **P < 0.01).

Article Snippet: Immunoblotting was performed after membrane blocking using rabbit polyclonal luc2 antibody (1:200; Fitzgerald Industries International, Acton, MA, USA).

Techniques: Staining, Expressing, Binding Assay, Activity Assay, Control, Transduction

Journal: Cell transplantation

Article Title: Optimized Longitudinal Monitoring of Stem Cell Grafts in Mouse Brain Using a Novel Bioluminescent/Near Infrared Fluorescent Fusion Reporter.

doi: 10.1177/0963689717739718

Figure Lengend Snippet: Fig. 5. Serial dilutions of luc2-iRFP720 expressing human mesenchymal stem cells (hMSCs) measured for bioluminescence and fluorescence intensity. (A) Bioluminescence imaging (BLI) was measured for the serial dilution of of luc2-iRFP720 expressing hMSCs (0, 3.125, 6.250, 12.500, 25.000, and 50.000 cells; r2 ¼ 0.9921). Data are represented as mean (SD) (n ¼ 3). (B) fluorescence imaging (FLI) was measured for the serial dilution (0, 3.125, 6.250, 12.500, 25.000, 50.000, and 100.000 cells) at 700 nm (r2 ¼ 0.9980) using the Odyssey scanner. Data are represented as mean (SD) (n ¼ 6). (C) BLI of decreased amount of cells (1 103, 1 104, 1 105, and 1 106). Images were taken 20 min after injection of D-luciferin (25 mM/kg). (D) FLI of decreased amounts of cells (1 103, 1 104, 1 105, and 1 106) when injected into the mouse brain using Pearl Imager with 700 nm settings.

Article Snippet: Immunoblotting was performed after membrane blocking using rabbit polyclonal luc2 antibody (1:200; Fitzgerald Industries International, Acton, MA, USA).

Techniques: Expressing, Fluorescence, Imaging, Serial Dilution, Injection

Journal: Cell transplantation

Article Title: Optimized Longitudinal Monitoring of Stem Cell Grafts in Mouse Brain Using a Novel Bioluminescent/Near Infrared Fluorescent Fusion Reporter.

doi: 10.1177/0963689717739718

Figure Lengend Snippet: Fig. 7. (A) Longitudinal monitoring of luc2-iRFP720 expressing human mesenchymal stem cells when injected in the mouse brain followed for 1, 3, 5, and 7 wk. (B) No significant differences in bioluminescence imaging of fluorescence imaging signal were observed between the weeks. Data are represented as mean (SD) (n ¼ 4; **P < 0.01 and *P < 0.05).

Article Snippet: Immunoblotting was performed after membrane blocking using rabbit polyclonal luc2 antibody (1:200; Fitzgerald Industries International, Acton, MA, USA).

Techniques: Expressing, Injection, Imaging, Fluorescence

Journal: British Journal of Pharmacology

Article Title: The nociceptin/orphanin FQ receptor system as a target to alleviate cancer‐induced bone pain in rats: Model validation and pharmacological evaluation

doi: 10.1111/bph.14899

Figure Lengend Snippet: Levels of inflammatory markers measured in plasma of sham and 1.5 × 10 6 ·ml −1 of MRMT‐1/Luc2‐bearing animals

Article Snippet: The rat mammary gland carcinoma cell line, MRMT‐1 (Tohoku University, JP; TKG Cat# TKG 0132, RRID:CVCL_5156), was transfected with the luciferase (Luc2) gene (Trenzyme, Life Science Services, DE) to allow monitoring of cell presence and tumour growth over time.

Techniques: Clinical Proteomics, Marker

Journal: British Journal of Pharmacology

Article Title: The nociceptin/orphanin FQ receptor system as a target to alleviate cancer‐induced bone pain in rats: Model validation and pharmacological evaluation

doi: 10.1111/bph.14899

Figure Lengend Snippet: Gene expression of oprl1 in dorsal root ganglion (DRG) and bone marrow, measured 21 days post‐surgery in sham and 1.5 × 10 6 ·ml −1 of MRMT‐1/Luc2‐bearing animals

Article Snippet: The rat mammary gland carcinoma cell line, MRMT‐1 (Tohoku University, JP; TKG Cat# TKG 0132, RRID:CVCL_5156), was transfected with the luciferase (Luc2) gene (Trenzyme, Life Science Services, DE) to allow monitoring of cell presence and tumour growth over time.

Techniques: Gene Expression

Journal: British Journal of Pharmacology

Article Title: The nociceptin/orphanin FQ receptor system as a target to alleviate cancer‐induced bone pain in rats: Model validation and pharmacological evaluation

doi: 10.1111/bph.14899

Figure Lengend Snippet: Effect of 1.25 × 10 6 ·ml −1 of MRMT‐1/Luc2 inoculated cells on (a) weight‐bearing ratio, (b) cold allodynia, and (c) mechanical allodynia, including the effect of i.p. morphine (3.16 mg·kg −1 ) administration. All data are presented as mean ± SEM; n = 9, all MRMT‐1/Luc2‐bearing animals; * P < .05 versus pretest

Article Snippet: The rat mammary gland carcinoma cell line, MRMT‐1 (Tohoku University, JP; TKG Cat# TKG 0132, RRID:CVCL_5156), was transfected with the luciferase (Luc2) gene (Trenzyme, Life Science Services, DE) to allow monitoring of cell presence and tumour growth over time.

Techniques:

Journal: British Journal of Pharmacology

Article Title: The nociceptin/orphanin FQ receptor system as a target to alleviate cancer‐induced bone pain in rats: Model validation and pharmacological evaluation

doi: 10.1111/bph.14899

Figure Lengend Snippet: (a) The effect of nociceptin (i.t.) and morphine (i.t.) and (b) the effect of systemic Ro65‐6570 (i.p.; NOP receptor agonist) and morphine (i.p.) on mechanical allodynia in 1.5 × 10 6 ·ml −1 of MRMT‐1/Luc2‐bearing animals in the von Frey test. (c) The effect of Ro65‐6570 (i.p.) is blocked by the NOP receptor antagonist J‐113397 (i.p.). All data are presented as mean ± SEM; n = 7 sham, n = 8 vehicle, n = 7 morphine, n = 8 nociceptin 1 μg, n = 8 nociceptin 3 μg, n = 6 nociceptin 10 μg in (a); n = 9 sham, n = 8 vehicle, n = 6 morphine, n = 7 Ro65‐6570 0.3 mg·kg −1 , n = 6 Ro65‐6570 1.0 mg·kg −1 , n = 5 Ro65‐6570 2.15 mg·kg −1 in (b); n = 9 vehicle, n = 8 Ro65‐6570, n = 8 J‐113397, n = 10 Ro65‐6570 + J‐113397 in (c); * P < .05 versus vehicle

Article Snippet: The rat mammary gland carcinoma cell line, MRMT‐1 (Tohoku University, JP; TKG Cat# TKG 0132, RRID:CVCL_5156), was transfected with the luciferase (Luc2) gene (Trenzyme, Life Science Services, DE) to allow monitoring of cell presence and tumour growth over time.

Techniques: