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

Abstract

The LacZ gene, which encodes Escherichia coli β-galactosidase, is widely used as a marker for cells with targeted gene expression or disruption. However, it has been difficult to detect lacZ-positive cells in living organisms or tissues at single-cell resolution, limiting the utility of existing lacZ reporters. Herein we present a newly developed fluorogenic β-galactosidase substrate suitable for labeling live cells in culture, as well as in living tissues. This precisely functionalized fluorescent probe exhibited dramatic activation of fluorescence upon reaction with the enzyme, remained inside cells by anchoring itself to intracellular proteins, and provided single-cell resolution. Neurons labeled with this probe preserved spontaneous firing, which was enhanced by application of ligands of receptors expressed in the cells, suggesting that this probe would be applicable to investigate functions of targeted cells in living tissues and organisms.

Treatment of diabetic mice with the SGLT2 inhibitor TA-1887 antagonizes diabetic cachexia and decreases mortality

“…Moreover, accumulation of senescent cells in eWAT, iWAT, and MG was confirmed by assessing senescence-associated beta-galactosidase activity using SPiDER beta-Gal staining. 21 Beta-Gal staining decreased in eWAT from TA-1887-treated relative to untreated mice, but was equivalent in iWAT and MG from treated and untreated mice (Fig. 3d ).…”

Red‐Shifted Fluorogenic Substrate for Detection of lacZ‐Positive Cells in Living Tissue with Single‐Cell Resolution

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The Escherichia coli lacZ gene encoding b-galactosidase is awidely used reporter,but few synthetic substrates are available for detecting its activity with single-cell resolution in living samples.O ur recently reported fluorogenic substrate SPiDER-bGal is suitable for this purpose,b ut its hydrolysis product shows green fluorescence emission, and ar ed-shifted analogue is therefore required for use in combination with green fluorescent protein (GFP) markers.Herein, we describe the development of ar ed-shifted fluorogenic substrate for bgalactosidase,S PiDER-Red-bGal, based on as ilicon rhodol scaffold and ac arboxylic group as the intramolecular nucleophile.L acZ-positive cells were successfully labeled with SPiDER-Red-bGal at single-cell resolution in living samples, which enabled us to visualized ifferent cell types in combination with GFP markers.The Escherichia coli lacZg ene,w hich encodes b-galactosidase,i sapowerful reporter in combination with synthetic substrates, [1] and many lacZ-expressing animals have been established for experimental use.H owever,a lthough several substrates are available for detecting b-galactosidase activity, [2] selective labeling of lacZ-positive cells with single-cell resolution in living tissues or organisms is not feasible with most existing probes.W er ecently developed an ew class of functionalized fluorogenic substrate for b-galactosidase, SPiDER-bGal, [3] which is suitable for this purpose;w e controlled the fluorescence through intramolecular spirocyclization and achieved cellular retention by application of quinone methide chemistry.However,the utility of SPiDER-bGal is limited because it emits green fluorescence at around 550 nm, and therefore it is difficult to use in combination with green fluorescent protein (GFP), [4] one of the most widely used fluorescent tags for biological studies.A lthough red fluorescent proteins have been widely used as reporters in the red wavelength region, their tendency to form aggregates when expressed in neurons in mouse brain may limit their applicability. [5] Therefore,d evelopment of ar ed fluorescent probe for b-galactosidase with single-cell resolution should extend the utility of the lacZreporter.Herein, we describe the development of an ew fluorogenic substrate for b-galactosidase,w hich emits red fluorescence at above 600 nm without loss of the advantageous properties of SPiDER-bGal.

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Proximity Ligation‐Based Fluorogenic Imaging Agents for Neuraminidases

“…These reagents allowed high-quality, imaging of enzymatic activity that was much more stable and discrete than that with the commonly used X-GlcA [10] . This concept has since been picked up by others and expanded to probe the biological activities of phosphatases [11] , sulfatases [12] , -lactamases [13] and -galactosidases [14] . In this paper, we describe our development of proximity ligation-based imaging agents for influenza virus neuraminidase in which a fluorogenic CF 2 Mu group, difluoromethyl coumarin, is attached to the anomeric position of sialic acid, CF 2 Mu-Neu5Ac, 1 .…”