Preparation of Single-cell Suspensions for Cytofluorimetric Analysis from Different Mouse Skin Regions

Broggi, Achille; Cigni, Clara; Zanoni, Ivan; Granucci, Francesca

doi:10.3791/52589

Cited by 15 publications

(8 citation statements)

References 15 publications

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“…An approximate 2-cm by 1-cm portion of skin was removed and dissociated with Liberase (Roche, 05401119001) according to a published protocol ( 63 ). The resulting cell suspension was incubated with anti-CD16/32 (BD553142) and Viability Dye eFlour780 (Invitrogen, 65-0865-14) in fluorescence-activated cell sorting (FACS) buffer containing 1% bovine serum albumin (BSA) and 2 mM EDTA in phosphate-buffered saline (PBS).…”

Section: Methodsmentioning

confidence: 99%

Dual antibody inhibition of KLK5 and KLK7 for Netherton syndrome and atopic dermatitis

Chavarría-Smith

Chiu²,

Jackman

et al. 2022

Sci. Transl. Med.

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The epidermis is a barrier that prevents water loss while keeping harmful substances from penetrating the host. The impermeable cornified layer of the stratum corneum is maintained by balancing continuous turnover driven by epidermal basal cell proliferation, suprabasal cell differentiation, and corneal shedding. The epidermal desquamation process is tightly regulated by balance of the activities of serine proteases of the Kallikrein-related peptidases (KLK) family and their cognate inhibitor lymphoepithelial Kazal type–related inhibitor (LEKTI), which is encoded by the serine peptidase inhibitor Kazal type 5 gene. Imbalance of proteolytic activity caused by a deficiency of LEKTI leads to excessive desquamation due to increased activities of KLK5, KLK7, and KLK14 and results in Netherton syndrome (NS), a debilitating condition with an unmet clinical need. Increased activity of KLKs may also be pathological in other dermatoses such as atopic dermatitis (AD). Here, we describe the discovery of inhibitory antibodies against murine KLK5 and KLK7 that could compensate for the deficiency of LEKTI in NS. These antibodies are protective in mouse models of NS and AD and, when combined, promote improved skin barrier integrity and reduced inflammation. To translate these findings, we engineered a humanized bispecific antibody capable of potent inhibition of human KLK5 and KLK7. A crystal structure of KLK5 bound to the inhibitory Fab revealed that the antibody binds distal to its active site and uses a relatively unappreciated allosteric inhibition mechanism. Treatment with the bispecific anti-KLK5/7 antibody represents a promising therapy for clinical development in NS and other inflammatory dermatoses.

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Section: Methodsmentioning

confidence: 99%

Dual antibody inhibition of KLK5 and KLK7 for Netherton syndrome and atopic dermatitis

Chavarría-Smith

Chiu²,

Jackman

et al. 2022

Sci. Transl. Med.

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“…Following transplantation with EMSC Sp (Envy), wound skin tissues were excised above, and dispersed into single-cell suspensions as previously described 67. In brief, the tissue was incubated with 0.5% Trypsin overnight at 4℃, minced completely, and incubated in a digestion buffer containing collagenase I (2.75 mg/ml), and DNase I (150 U/ml) in a 37℃ shaking bath for 2 h. The trypsin digests were pooled and filtered through a nylon cell strainer with the pore size at 40 μm.…”

Section: Methodsmentioning

confidence: 99%

Noninvasive application of mesenchymal stem cell spheres derived from hESC accelerates wound healing in a CXCL12-CXCR4 axis-dependent manner

Wang¹,

Jiang²,

Sun³

et al. 2019

Theranostics

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Mesenchymal stem cells (MSC) derived from adult tissues effectively promote wound healing. However, MSC quality varies, and the quantity of MSC is limited, as MSC are acquired through donations. Moreover, the survival and functioning of dissociated MSC delivered to an inflammatory lesion are subject to challenges.Methods: Here, spheres (EMSCSp) generated from human embryonic stem cell-derived MSC (EMSC) were directly dropped onto excised wounds in mice; the effects of EMSCSp were compared to those of dissociated EMSC (EMSCDiss). Following transplantation, we measured the extent of wound closure, dissected the histological features of the wounds, determined transcriptomic changes in cells isolated from the treated and control wounds, and evaluated the molecular mechanism of the effects of EMSC.Results: The application of EMSCSp onto murine dermal wounds substantially increased survival and efficacy of EMSC compared to the topical application of EMSCDiss. RNA sequencing (RNA-Seq) of cells isolated from the wounds highlighted the involvement of CXCL12-CXCR4 signaling in the effects of EMSCSp, which was verified in EMSC via CXCL12 knockdown and in target cells (vascular endothelial cells, epithelial keratinocytes, and macrophages) via CXCR4 inhibition. Finally, we enhanced the biosafety of EMSCSp by engineering cells with an inducible suicide gene.Conclusions: Together, these data suggest the topical application of EMSCSp as an unlimited, quality-assured, safe, and noninvasive therapy for wound healing and the CXCL12-CXCR4 axis as a key player in this treatment.

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“…Flow cytometry. The single-cell suspensions of mouse ears were prepared based on the previous method 57,58 . Briefly, the ears were split and cut, incubated with a solution of RPMI containing 1 mg/ml DNase I (NIPPON GENE, Tokyo, Japan) and 1 mg/ml collagenase (Worthington Biochemical, Lakewood, NJ, USA) 90 min at 37 °C.…”

Section: Methodsmentioning

confidence: 99%

Semaphorin 3A: A potential target for prevention and treatment of nickel allergy

et al. 2022

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Metal allergy is one of the typical immune disorders encountered during the application of dental/medical materials and has a highly complex pathogenic mechanism. Semaphorin 3A (Sema3A), a member of the semaphorin family, is reported to be involved in various immune disorders. However, its role in metal allergy has not been clarified yet. Herein, we show that Sema3A expression was upregulated in nickel (Ni) allergy-induced mouse ear tissue and in NiCl2-stimulated mouse keratinocytes. Moreover, Sema3A regulated tumor necrosis factor-alpha production and mitogen-activated protein kinase activation in keratinocytes. The specific deletion of Sema3A in keratinocytes did not affect immune cell infiltration but reduced edema and ear swelling; it also impeded Th1 responses to cause a slight alleviation in Ni allergy in mice. Our results demonstrate that Sema3A promotes the development of metal allergy and should be explored as a potential target for the prevention and treatment of metal allergy.

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Preparation of Single-cell Suspensions for Cytofluorimetric Analysis from Different Mouse Skin Regions

Cited by 15 publications

References 15 publications

Dual antibody inhibition of KLK5 and KLK7 for Netherton syndrome and atopic dermatitis

Dual antibody inhibition of KLK5 and KLK7 for Netherton syndrome and atopic dermatitis

Noninvasive application of mesenchymal stem cell spheres derived from hESC accelerates wound healing in a CXCL12-CXCR4 axis-dependent manner

Semaphorin 3A: A potential target for prevention and treatment of nickel allergy

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