Detection of <i>LacZ</i>‐Positive Cells in Living Tissue with Single‐Cell Resolution

Doura, Tomohiro; Kamiya, Mako; Obata, Fumiaki; Yamaguchi, Yoshifumi; Hiyama, Takeshi Y.; Matsuda, Takashi; Fukamizu, Akiyoshi; Noda, Masaharu; Miura, Masayuki; Urano, Yasuteru

doi:10.1002/ange.201603328

Cited by 37 publications

(39 citation statements)

References 22 publications

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“…We then examined the expression of C2cd4c in the pancreas by visualizing LacZ activity. For this, we used Salmon‐gal (S‐gal; 6‐chloro‐3‐indolyl‐beta‐ d ‐galactopyranoside) in combination with Nitro Blue tetrazolium , or SPiDER‐βGal, which is rendered fluorescent by the enzymatic reaction . We visualized C2cd4c / LacZ activity during embryonic development (at E14.5–18.5) and in adult C2cd4c / LacZ KI heterozygous mice (Fig.…”

Section: Resultsmentioning

confidence: 99%

Changes in expression of C2cd4c in pancreatic endocrine cells during pancreatic development

et al. 2016

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C2cd4c, encoded by a gene belonging to the C2cd4 family, contains a C2 domain conserved across species and is localized to the cytoplasm. To examine the role of C2cd4c in the pancreas, we studied its localization and generated C2cd4c knockout (KO) mice. C2cd4c was expressed in pancreatic endocrine progenitors at early embryonic stages. When endocrine cells arise from their precursors, C2cd4c is gradually confined to the insulin‐ and pancreatic polypeptide‐expressing cells of the endocrine. In the adult pancreas, C2cd4c is restricted to the beta cells. C2cd4c KO mice showed normal embryonic pancreatic development and adult pancreatic function. Thus, our results suggest that C2cd4c is dispensable for pancreatic development.

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Section: Resultsmentioning

confidence: 99%

Changes in expression of C2cd4c in pancreatic endocrine cells during pancreatic development

et al. 2016

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“…To sort senescent cells by flow cytometry, the cells were isolated from mouse corneal stroma with collagenase treatment and labeled with the cellular senescence detection kit SPiDERb-gal (Dojindo Lab, Kumamoto, Japan), as described previously. 42,43 Briefly, SPiDER-b-gal was added and incubated with the cells for 30 minutes at 378C under protection from light. After incubation, the cells were colabeled with the fibroblast marker vimentin-Alexa Fluor 647 (Abcam), washed, and sorted immediately using a FACScan flow cytometer (BD FACSAria3; BD Biosciences, San Jose, CA, USA) for SPiDER-b-galþ and vimentinþ cells.…”

Section: Flow Cytometry Assaymentioning

confidence: 99%

Induction of Fibroblast Senescence During Mouse Corneal Wound Healing

Wang

et al. 2019

Invest. Ophthalmol. Vis. Sci.

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Induction of fibroblast senescence during mouse corneal wound healing. Invest Ophthalmol Vis Sci. 2019;60:3669-3679. https://doi.org/10.1167/iovs.19-26983 PURPOSE. To investigate the presence and role of fibroblast senescence in the dynamic process of corneal wound healing involving stromal cell apoptosis, proliferation, and differentiation.METHODS. An in vivo corneal wound healing model was performed using epithelial debridement in C57BL/6 mice. The corneas were stained using TUNEL, Ki67, and a-smooth muscle actin (a-SMA) as markers of apoptosis, proliferation, and myofibroblastic differentiation, respectively. Cellular senescence was confirmed by senescence-associated bgalactosidase (SA-b-gal) staining and P16 Ink4a expression. Mitogenic response and gene expression were compared among normal fibroblasts, H 2 O 2 -induced senescent fibroblasts, and TGF-b-induced myofibroblasts in vitro. The senescence was further detected in mouse models of corneal scarring, alkali burn, and penetrating keratoplasty (PKP). RESULTS.The apoptosis and proliferation of corneal stromal cells were found to peak at 4 and 24 hours after epithelial debridement. Positive staining of SA-b-gal was observed clearly in the anterior stromal cells at 3 to 5 days. The senescent cells displayed P16 Ink4a þ vimentinþ a-SMAþ, representing the major origin of activated corneal resident fibroblasts. Compared with normal fibroblasts and TGF-b-induced myofibroblasts, H 2 O 2 -induced senescent fibroblasts showed a nonfibrogenic phenotype, including a reduced response to growth factor basic fibroblast growth factor (bFGF) or platelet-derived growth factor-BB (PDGF-BB), increased matrix metalloproteinase (MMP)1/3/13 expression, and decreased fibronectin and collagen I expression. Moreover, cellular senescence was commonly found in the mouse corneal scarring, alkali burn, and PKP models. CONCLUSIONS.Corneal epithelial debridement induced the senescence of corneal fibroblasts after apoptosis and proliferation. The senescent cells displayed a nonfibrogenic phenotype and may be involved in the self-limitation of corneal fibrosis.

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“…Moreover, fluorescence signal increased gradually even after removal of SPiDER-βGal. Doura et al reported that fluorescence derived from SPiDER-βGal increased with time until 120 min, the end of observation, in vitro [ 14 ], suggesting that it happens slowly in a few hours to form the protein anchored molecule from SPiDER-βGal. Thus, we speculated that SPiDER-βGal which remained inside of cell was activated by β-galactosidase and bound to intracellular protein gradually, leading to a progressive increase in fluorescence intensity even after removal of SPiDER-βGal.…”

Section: Discussionmentioning

confidence: 99%

“…SPiDER-βGal was obtained from Dojindo Molecular Technologies, Inc. (Rockville, MD, USA) [ 14 ]. gGlu-HMRG, a GGT activated fluorescence probe was synthesized as described previously [ 3 ].…”

Section: Methodsmentioning

confidence: 99%

“…SPiDER-βGal is a newly developed fluorogenic β-galactosidase substrate suitable for labeling live cells in culture, as well as in living tissues. In contrast to the first generation agent, this fluorescent probe exhibited dramatic activation of fluorescence upon reaction with β-galactosidase, which persisted as the probe internalized and was anchored to intracellular proteins, enabling single cell resolution [ 14 ]. Its capacity to internalize, bind and be retained intracellularly likely accounts for the persistence of fluorescence over time.…”

Section: Introductionmentioning

confidence: 99%

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A topically-sprayable, activatable fluorescent and retaining probe, SPiDER-βGal for detecting cancer: Advantages of anchoring to cellular proteins after activation

et al. 2017

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SPiDER-βGal is a newly-developed probe that is activated by β-galactosidase and is then retained within cells by anchoring to intracellular proteins. Previous work has focused on gGlu-HMRG, a probe activated by γ-glutamyltranspeptidase, which demonstrated high sensitivity for the detection of peritoneal ovarian cancer metastases in an animal model. However, its fluorescence, after activation by γ-glutamyltranspeptidase, rapidly declines over time, limiting the actual imaging window and the ability to define the border of lesions. The purpose of this study is to compare the fluorescence signal kinetics of SPiDER-βGal with that of gGlu-HMRG using ovarian cancer cell lines in vitro and ex vivo tissue imaging. In vitro removal of gGlu-HMRG resulted in a rapid decrease of fluorescence intensity followed by a more gradual decrease up to 60 min while there was a gradual increase in fluorescence up to 60 min after removal of SPiDER-βGal. This is most likely due to internalization and retention of the dye within cells. This was also confirmed ex vivo tissue imaging using a red fluorescence protein (RFP)-labeled tumor model in which the intensity of fluorescence increased gradually after activation of SPiDER-βGal. Additionally, SPiDER-βGal resulted in intense enhancement within the tumor due to the high target-to-background ratio, which extended up to 60 min after activation. In contrast, gGlu-HMRG fluorescence resulted in decreasing fluorescence over time in extracted tumors. Thus, SPiDER-βGal has the advantages of higher signal with more signal retention, resulting in improved contrast of the tumor margin and suggesting it may be an alternative to existing activatable probes.

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Detection of LacZ‐Positive Cells in Living Tissue with Single‐Cell Resolution

Cited by 37 publications

References 22 publications

Changes in expression of C2cd4c in pancreatic endocrine cells during pancreatic development

Changes in expression of C2cd4c in pancreatic endocrine cells during pancreatic development

Induction of Fibroblast Senescence During Mouse Corneal Wound Healing

A topically-sprayable, activatable fluorescent and retaining probe, SPiDER-βGal for detecting cancer: Advantages of anchoring to cellular proteins after activation

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