一种基于图像的动态高通量分析黏附细胞的迁移

Meng Sun; Bence  Rethi; Akilan Krishnamurthy; Vijay Joshua; Heidi Wähämaa; Sergiu-Bogdan Catrina; Anca Catrina

doi:10.21769/BioProtoc.3957

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Peer-reviewed

An Image-based Dynamic High-throughput Analysis of Adherent Cell Migration

一种基于图像的动态高通量分析黏附细胞的迁移

MS Meng Sun email

BR Bence Rethi

AK Akilan Krishnamurthy

VJ Vijay Joshua

HW Heidi Wähämaa

SC Sergiu-Bogdan Catrina

AC Anca Catrina email

发布: 2021年03月20日第11卷第6期 DOI: 10.21769/BioProtoc.3957 浏览次数: 5172

评审: Pedro EscollAnonymous reviewer(s)

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The authors used this protocol in:

Cover of Annals of the Rheumatic Diseases, featuring study using the protocol.

Dec 2019

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Abstract

In this protocol, we describe a method to monitor cell migration by live-cell imaging of adherent cells. Scratching assay is a common method to investigate cell migration or wound healing capacity. However, achieving homogenous scratching, finding the optimal time window for end-point analysis and performing an objective image analysis imply, even for practiced and adept experimenters, a high chance for variability and limited reproducibility. Therefore, our protocol implemented the assessment for cell mobility by using homogenous wound making, sequential imaging and automated image analysis. Cells were cultured in 96-well plates, and after attachment, homogeneous linear scratches were made using the IncuCyte^® WoundMaker. The treatments were added directly to wells and images were captured every 2 hours automatically. Thereafter, the images were processed by defining a scratching mask and a cell confluence mask using a software algorithm. Data analysis was performed using the IncuCyte^® Cell Migration Analysis Software. Thus, our protocol allows a time-lapse analysis of treatment effects on cell migration in a highly reliable, reproducible and re-analyzable manner.

Keywords: Cell migration (细胞迁移)

Scratching assay (划痕法)

Live cell image (活细胞成像)

Time-lapse imaging (延时成像)

High-throughput (高通量)

Background

Scratching assays are a widely used method for investigating cell migration or wound healing capacity. However, the conventional method (manual scratching) requires skill to perform linear scratches and is an end-point assay (Liang et al., 2007; Krishnamurthy et al., 2016). Data are usually manually analyzed with ImageJ or other software. Recently, we employed a high-throughput automatic imaging system, IncuCyte ZOOM from Essen Bioscience, in a cell migration assay (Sun et al., 2019). By using IncuCyte^® WoundMaker, linear scratches can be created homogeneously in up to 96-wells at the same time. With the appropriately defined algorithm, by analysis of phase-contrast, cell confluence masks and scratching masks, cell migration can be simultaneously evaluated. In brief, the conventional method is more laborious and time-consuming than the method we present here. This protocol provides a method with minimized time and effort for processing high-throughput samples and analyzing data in an unbiased way over time.

Materials and Reagents

IncuCyte^® ImageLock 96-well Plates (Essen Bioscience, catalog number: 4379 )
Synovial fibroblast (Isolated from RA patients undergoing joint replacement, Sun et al., 2019)
Normal human dermal fibroblasts (PromoCell, catalog number: C-12300 )
Primary Human Osteoarthritis Synovial Fibroblasts (Bioivit, catalog number: HPCSFOA-03 )
Dulbecco's Modified Eagle Medium (DMEM) (Sigma-Aldrich, catalog number: D5796-500ml )
Fetal bovine serum (FBS) (Sigma-Aldrich, catalog number: F7524 )
Trypsin-EDTA (Sigma-Aldrich, catalog number: T3924-100ml )
Phosphate buffered saline (PBS) (Sigma-Aldrich, catalog number: D8537-500ml )
Penicillin-streptomycin (PEST) (Sigma-Aldrich, catalog number: P4333-100ml )
Anti-citrullinated protein antibody (Purified from peripheral blood of RA patients, Ossipova et al., 2014)
Recombinant Human TNF-α (Peprotech, catalog number: 300-01A )
Recombinant Human IL-8/CXCL8 Protein (R&D Systems, catalog number: 208-IL-010 )
Alconox powder (VWR, catalog number: 21835-123 )
Sachets, Rely+On^TM Virkon^® powder (VWR, catalog number: 148-0200 )
Sterile distilled water (produced in house)
70% ethanol ( Sigma-Aldrich, catalog number: 470198-1L )
Synovial fibroblasts culture medium (10% FBS) (see Recipes)
Starvation medium (serum free) (see Recipes)
Low-serum cell culture medium (2% FBS) (see Recipes)

Equipment

IncuCyte^® WoundMaker with two wash boats (Essen Bioscience, catalog number: 4493 )
IncuCyte ZOOM live-cell analysis system (Essen Bioscience, model: IncuCyte^® ZOOM )
Multi-Channel pipette, 8-channel, 20-200 μl (VWR, Ergonomic High Performance Multichannel Pipettor, catalog number: 89079-948 )

Software

Cell Migration Analysis Software Module (Essen Bioscience, catalog number: 4400)
Prism 6 (GraphPad Software)
IncuCyte^® Zoom software (2018A)

Procedure

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Sun, M., Rethi, B., krishnamurthy, A., Joshua, V., Wähämaa, H., Catrina, S. and Catrina, A. (2021). An Image-based Dynamic High-throughput Analysis of Adherent Cell Migration. Bio-protocol 11(6): e3957. DOI: 10.21769/BioProtoc.3957.