Using High Content Imaging to Quantify Target Engagement in Adherent Cells

Hanna Axelsson; Helena Almqvist; Brinton Seashore-Ludlow

doi:10.3791/58670

JoVE Journal
Biochemistry

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JoVE Journal Biochemistry

Using High Content Imaging to Quantify Target Engagement in Adherent Cells

척도 부착 셀에 대상 참여를 이미징 높은 콘텐츠를 사용 하 여

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07:23 min

November 29, 2018

DOI: 10.3791/58670-v

Hanna Axelsson^1,2, Helena Almqvist^1,2, Brinton Seashore-Ludlow^1,3

¹Chemical Biology Consortium Sweden, Science for Life Laboratory,Karolinska Institutet, Solna, ²Department of Medical Biochemistry and Biophysics,Karolinska Institutet, Solna, ³Science for Life Laboratory, Department of Oncology-Pathology,Karolinska Institutet, Stockholm

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Overview

This article details a protocol for measuring drug-target engagement using high content imaging in a microplate-compatible adaptation of the cellular thermal shift assay (CETSA). This method is crucial for answering key questions in chemical biology and drug discovery.

Key Study Components

Area of Science

Chemical Biology
Drug Discovery
Imaging Techniques

Background

Drug target engagement is essential for effective drug development.
The cellular thermal shift assay (CETSA) is a valuable tool in this context.
This method allows for spatial localization through imaging.
It can be applied to various cell systems, including primary cultures and cocultures.

Purpose of Study

To determine if a compound binds to the target protein.
To validate chemical probes in drug development.
To enhance understanding of drug-target interactions.

Methods Used

High content imaging in microplate format.
Preparation of black 384-well imaging assay plates.
Use of aluminum foil to cover plates during cell seeding.
Drilling holes in plates for proper handling.

Main Results

The method allows for effective measurement of drug-target engagement.
It provides spatial localization of drug interactions.
Applicable to various cell types beyond cell lines.
Facilitates the validation of chemical probes.

Conclusions

This protocol is a significant advancement in drug discovery techniques.
It offers a reliable method for assessing drug-target interactions.
Future applications may extend to more complex biological systems.

Frequently Asked Questions

What is the cellular thermal shift assay (CETSA)?

CETSA is a method used to measure drug-target engagement by assessing the thermal stability of target proteins in the presence of compounds.

Can this method be used with primary cultures?

Yes, the method is applicable to primary cultures and cocultures, in addition to standard cell lines.

What are the advantages of using high content imaging?

High content imaging allows for detailed spatial localization of drug interactions without detaching adherent cells.

How are the assay plates prepared?

The plates are covered with aluminum foil and drilled with holes for proper handling before cell seeding.

What key questions does this method help answer?

It helps determine whether a compound binds to a target protein, which is crucial for drug development.

Is this method suitable for high-throughput screening?

Yes, the microplate-compatible format makes it suitable for high-throughput screening applications.

마약 대상 포용의 측정은 효과적인 약물 개발 및 화학 프로브 유효성 검사를 중앙. 여기, 우리 선발 마약 대상 포용 셀룰러 열 교대 분석 실험 (CETSA)의 미 판 호환 adaption에 높은 콘텐츠 이미징 사용 하 여 측정 하기 위한 프로토콜.

이 방법은 화학 생물학 및 약물 발견의 주요 질문에 대답하는 데 도움이 될 수 있습니다. 이러한 화합물은 표적 단백질에 결합합니까? 이 기술의 주요 장점은 부착 된 세포의 분리 요구 사항이 없다는 것입니다.

그리고 공간 국소화는 이미징에 의해 결정될 수 있다. 우리는 세포주에서 이 방법을 적용했습니다, 1 차적인 배양 및 동배와 같은 그밖 세포 시스템에 이 방법을 적용하는 것은 또한 가능합니다. 세포를 파종하기 전에, 조직 배양 후드에 검은 색 384 웰 이미징 분석 플레이트를 배치하고, 각 플레이트의 양쪽 끝에 세 개의 3.5 밀리미터 직경 구멍을 만들기 위해 표준 드릴을 사용하기 전에 알루미늄 호일로 플레이트를 덮어.

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