Protocol for Primary Mouse Hepatocyte Isolation

Charni-Natan, Meital; Goldstein, Ido

doi:10.1016/j.xpro.2020.100086

Cited by 223 publications

(126 citation statements)

References 13 publications

(9 reference statements)

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“…The protocol for preparing non-parenchymal cells was optimized from several other available protocols ( Mederacke et al., 2015 ; Movita et al., 2012 ; Seki et al., 2009 ). Excellent protocols with videos detailing the procedure for perfusion-based digestion of a mouse liver exist and can be referred to ( Charni-Natan and Goldstein, 2020 ; Mederacke et al., 2015 ). Note: The protocol benefits from two people working in parallel if processing more than four mice.…”

Section: Step-by-step Methods Detailsmentioning

confidence: 99%

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Purification of mouse hepatic non-parenchymal cells or nuclei for use in ChIP-seq and other next-generation sequencing approaches

et al. 2021

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Summary Significant advancements in understanding disease mechanisms can occur through combined analysis of next-generation sequencing datasets generated using purified cell populations. Here, we detail our optimized protocol for purification of mouse hepatic macrophages (or other liver non-parenchymal populations) suitable for use in various next-generation sequencing protocols. An alternative framework is described for sorting pre-fixed hepatic nuclei populations. This strategy has the advantage of rapidly preserving the nuclei and can facilitate success with ChIP-seq for more challenging molecules. For complete details on the use and execution of these protocols, please refer to Muse et al. (2018) , Sakai et al. (2019) , and Seidman et al. (2020) .

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Section: Step-by-step Methods Detailsmentioning

confidence: 99%

“…Thorough descriptions of strategies to improve liver digestions have already been described elsewhere. ( Charni-Natan and Goldstein, 2020 ; Mederacke et al., 2015 ).…”

Section: Troubleshootingmentioning

confidence: 99%

Purification of mouse hepatic non-parenchymal cells or nuclei for use in ChIP-seq and other next-generation sequencing approaches

et al. 2021

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“…Primary mice hepatocytes were isolated based on the method described previously ( Charni-Natan and Goldstein, 2020 ). In brief, rat liver was routinely perfused with D-Hank’s (calcium free) and heparin for about 15 min followed by digestion by using 0.5 % collagenase solution supplemented with calcium for 30 min.…”

Section: Methodsmentioning

confidence: 99%

SAA1/TLR2 axis directs chemotactic migration of hepatic stellate cells responding to injury

et al. 2021

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Summary Hepatic stellate cells (HSCs) are crucial for liver injury repair and cirrhosis. However, the mechanism of chemotactic recruitment of HSCs into injury loci is still largely unknown. Here, we demonstrate that serum amyloid A1 (SAA1) acts as a chemokine recruiting HSCs toward injury loci signaling via TLR2, a finding proven by gene manipulation studies in cell and mice models. The mechanistic investigations revealed that SAA1/TLR2 axis stimulates the Rac GTPases through PI3K-dependent pathways and induces phosphorylation of MLC (pSer19). Genetic deletion of TLR2 and pharmacological inhibition of PI3K diminished the phosphorylation of MLCpSer19 and migration of HSCs. In brief, SAA1 serves as a hepatic endogenous chemokine for the TLR2 receptor on HSCs, thereby initiating PI3K-dependent signaling and its effector, Rac GTPases, which consequently regulates actin filament remodeling and cell directional migration. Our findings provide novel targets for anti-fibrosis drug development.

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“… Note: Primary hepatocytes is an important physiological model of the liver in vitro, which has been widely used in the biomedical field ( Bantubungi et al., 2014 ; Charni-Natan and Goldstein, 2020 ; Salem et al, 2018 ). The detailed preparation is as follows.…”

Section: Step-by-step Methods Detailsmentioning

confidence: 99%

Protocol for visualizing newly synthesized proteins in primary mouse hepatocytes

et al. 2021

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Summary Selective identification of newly synthesized proteins is challenging because all proteins, both existing and nascent, have the same amino acid pool and are therefore chemically indistinguishable. L-homopropargylglycine is an amino acid analog of methionine containing an alkyne moiety that can undergo a classic click chemical reaction with azide containing Alexa Fluor. Here, we present an integrated tool based on immunofluorescence staining to accurately trace and localize the newly synthesized protein in isolated primary mouse hepatocytes. For complete details on the use and execution of this protocol, please refer to Shen et al. (2021) .

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Protocol for Primary Mouse Hepatocyte Isolation

Cited by 223 publications

References 13 publications

Purification of mouse hepatic non-parenchymal cells or nuclei for use in ChIP-seq and other next-generation sequencing approaches

Purification of mouse hepatic non-parenchymal cells or nuclei for use in ChIP-seq and other next-generation sequencing approaches

SAA1/TLR2 axis directs chemotactic migration of hepatic stellate cells responding to injury

Protocol for visualizing newly synthesized proteins in primary mouse hepatocytes

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