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JoVE Journal
Immunology and Infection
Enzimatik Digest ve Gradient Ayırma kullanma Fare Skin gelen sızan lökositlerin İzolasyonu
Enzimatik Digest ve Gradient Ayırma kullanma Fare Skin gelen sızan lökositlerin İzolasyonu
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

Enzimatik Digest ve Gradient Ayırma kullanma Fare Skin gelen sızan lökositlerin İzolasyonu

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.

Bu protokol, fare derisinin besin açısından zengin ortamda enzimatik sindirimini ve ardından lökositleri izole etmek için gradyan ayrımını tanımlar. Bu şekilde türetilen hücreler, çeşitli alt uygulamalar için kullanılabilir. Bu, doku ayrışma makinelerine ve daha sert tripsin ve dispas bazlı doku sindirim protokollerine etkili, ekonomik ve gelişmiş bir alternatiftir.

Bu prosedürün genel amacı, enzimatik sindirim ve yoğunluk gradyan ayrımı kullanarak cilde sızan lökositleri izole etmektir. Bu yöntem, atopik dermatit de dahil olmak üzere cilt patolojilerinin birçok fare modelinde hücresel ve moleküler mekanizmaların araştırılmasına uygulanabilir. Bu yöntem cilde sızan lökosit biyolojisi hakkında bilgi sağlayabilse de, diğer yerleşik hücre popülasyonlarının araştırılmasına da uygulanabilir.

Genel olarak, bu yönteme yeni başlayan kişiler, fazla yağ ve bağ dokularının tamamen çıkarılmasını içeren deri toplama işlemi ile mücadele edeceklerdir. Bu yöntemin görsel olarak gösterilmesi kritik öneme sahiptir, çünkü deri toplama ve yoğunluk gradyanı işlemenin öğrenilmesi zordur ve hassasiyet ve pratik gerektirir. Tedaviden sonra uygun zaman noktasında, sekiz ila on iki haftalık yetişkin bir dişi ND4 Swiss-Webster faresinden 25 x 25 milimetrelik traşlı yan deri alanını dikkatlice çıkarmak için 10 santimetre cerrahi makas kullanın.

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