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JoVE Journal
Immunology and Infection
从小鼠皮肤使用酶消化和梯度分离提取白细胞浸润的
从小鼠皮肤使用酶消化和梯度分离提取白细胞浸润的
JoVE Journal
Immunology and Infection
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JoVE Journal Immunology and Infection
Isolation of Infiltrating Leukocytes from Mouse Skin Using Enzymatic Digest and Gradient Separation

从小鼠皮肤使用酶消化和梯度分离提取白细胞浸润的

Full Text
22,322 Views
07:11 min
January 25, 2016

DOI: 10.3791/53638-v

Charles J. Benck1, Tijana Martinov2, Brian T. Fife2, Devavani Chatterjea1

1Department of Biology,Macalester College, 2Department of Medicine, Division of Rheumatic and Autoimmune Diseases, Center for Immunology,University of Minnesota

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Please note that some of the translations on this page are AI generated. Click here for the English version.

Overview

This protocol describes the isolation of skin-infiltrating leukocytes using enzymatic digestion and density gradient separation. This method is effective for studying cellular and molecular mechanisms in mouse models of skin pathologies, such as atopic dermatitis.

Key Study Components

Area of Science

  • Neuroscience
  • Cell Biology
  • Immunology

Background

  • Skin-infiltrating leukocytes play a crucial role in skin pathologies.
  • Traditional methods for tissue dissociation can be harsh and less effective.
  • Enzymatic digestion offers a more economical alternative.
  • Precision is required in skin harvesting and gradient handling.

Purpose of Study

  • To isolate leukocytes from mouse skin for further analysis.
  • To provide insights into skin-infiltrating leukocyte biology.
  • To facilitate the study of other resident cell populations.

Methods Used

  • Enzymatic digestion of mouse skin in nutrient-rich medium.
  • Gradient separation to isolate leukocytes.
  • Careful excision of skin using surgical scissors.
  • Visual demonstration to aid in learning the technique.

Main Results

  • Successful isolation of leukocytes from mouse skin.
  • Improved understanding of skin-infiltrating leukocyte biology.
  • Potential applications in studying various skin pathologies.
  • Demonstrated effectiveness of the method compared to traditional techniques.

Conclusions

  • The enzymatic digestion and gradient separation method is effective and economical.
  • This protocol can enhance research on skin-related diseases.
  • Visual aids are essential for mastering the technique.

Frequently Asked Questions

What is the main goal of this protocol?
The main goal is to isolate skin-infiltrating leukocytes for further study.
Why is visual demonstration important?
Visual demonstration helps new users learn the precise techniques required for skin harvesting and gradient separation.
What are the advantages of this method?
This method is effective, economical, and provides an improved alternative to harsher tissue dissociation techniques.
Can this method be applied to other cell populations?
Yes, it can also be used to investigate other resident cell populations in the skin.
What type of mouse is used in this protocol?
The protocol uses adult eight to twelve-week-old female ND4 Swiss-Webster mice.
What tools are necessary for the procedure?
10 centimeter surgical scissors are required for excising the skin sample.

该方案描述了在营养丰富的培养基中对小鼠皮肤进行酶消化,然后进行梯度分离以分离白细胞。 由此衍生的电解槽可用于各种下游应用。这是组织解离机和更苛刻的基于胰蛋白酶和分散酶的组织消化方案的有效、经济且改进的替代方案。

该程序的总体目标是使用酶消化和密度梯度分离分离皮肤浸润的白细胞。该方法可应用于许多皮肤病理(包括特应性皮炎)小鼠模型中的细胞和分子机制的研究。虽然这种方法可以提供对皮肤浸润白细胞生物学的见解,但它也可以应用于其他常驻细胞群的研究。

一般来说,刚接触这种方法的人会努力收集皮肤,其中包括完全去除多余的脂肪和结缔组织。这种方法的视觉演示至关重要,因为皮肤采集和密度梯度处理很难学习,并且需要精确和练习。在治疗后的适当时间点,使用 10 厘米手术剪刀小心地从 8 至 12 周龄的成年雌性 ND4 Swiss-Webster 小鼠身上切除 25 x 25 毫米的剃光侧腹皮肤区域。

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