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Dec 2019
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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   

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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

  1. IncuCyte® ImageLock 96-well Plates (Essen Bioscience, catalog number: 4379 )

  2. Synovial fibroblast (Isolated from RA patients undergoing joint replacement, Sun et al., 2019)

  3. Normal human dermal fibroblasts (PromoCell, catalog number: C-12300 )

  4. Primary Human Osteoarthritis Synovial Fibroblasts (Bioivit, catalog number: HPCSFOA-03 )

  5. Dulbecco's Modified Eagle Medium (DMEM) (Sigma-Aldrich, catalog number: D5796-500ml )

  6. Fetal bovine serum (FBS) (Sigma-Aldrich, catalog number: F7524 )

  7. Trypsin-EDTA (Sigma-Aldrich, catalog number: T3924-100ml )

  8. Phosphate buffered saline (PBS) (Sigma-Aldrich, catalog number: D8537-500ml )

  9. Penicillin-streptomycin (PEST) (Sigma-Aldrich, catalog number: P4333-100ml )

  10. Anti-citrullinated protein antibody (Purified from peripheral blood of RA patients, Ossipova et al., 2014)

  11. Recombinant Human TNF-α (Peprotech, catalog number: 300-01A )

  12. Recombinant Human IL-8/CXCL8 Protein (R&D Systems, catalog number: 208-IL-010 )

  13. Alconox powder (VWR, catalog number: 21835-123 )

  14. Sachets, Rely+OnTM Virkon® powder (VWR, catalog number: 148-0200 )

  15. Sterile distilled water (produced in house)

  16. 70% ethanol ( Sigma-Aldrich, catalog number: 470198-1L )

  17. Synovial fibroblasts culture medium (10% FBS) (see Recipes)

  18. Starvation medium (serum free) (see Recipes)

  19. Low-serum cell culture medium (2% FBS) (see Recipes)

Equipment

  1. IncuCyte® WoundMaker with two wash boats (Essen Bioscience, catalog number: 4493 )

  2. IncuCyte ZOOM live-cell analysis system (Essen Bioscience, model: IncuCyte® ZOOM )

  3. Multi-Channel pipette, 8-channel, 20-200 μl (VWR, Ergonomic High Performance Multichannel Pipettor, catalog number: 89079-948 )

Software

  1. Cell Migration Analysis Software Module (Essen Bioscience, catalog number: 4400)

  2. Prism 6 (GraphPad Software)

  3. IncuCyte® Zoom software (2018A)

Procedure

  1. Prepare Cells

    1. Seed Cells in 150 μl complete growth medium in a 96-well imagelock plate using a multi-channel pipette at a cell density that will reach up to 95% confluence overnight. In 96-well plates, it is advisable to exclude the outer wells from the experiment due to evaporation effects.

      Note: We seed 20,000 synovial fibroblasts or human dermal fibroblasts per well to reach full confluence within 24 h.

    2. Fill the outer wells with 300 μl PBS to counteract evaporation-effects in rest of the wells.

    3. Grow cells at 37°C in a humidified incubator with 5% CO2 overnight or until cells reach 95% confluence.

    4. Wash cells with 100 μl PBS twice using a multi-channel pipette.

    5. Starve cells with 100 μl FBS free culture medium in a humidified incubator with 5% CO2 for 2 h to deplete growth factors.

      Note: In our setting, we starve cells for 2 h. Overnight starvation is commonly used for growth factor depletion. Starvation time may differ, depending on the experimental setting.


  2. Make scratch

    1. Clean the wound maker in the wash boat for 5 minutes each in a series of four wash solutions (45 ml of each)

      1. 0.5% Alconox

      2. 1% Virkon

      3. sterile distilled water

      4. 70% ethanol

    2. Use the IncuCyte® Wound Maker to create homogenous scratches

      (https://www.youtube.com/watch?v=x7pMzJ1VIdA&feature=youtu.be)

      1. Remove top of the wound maker and place it in an empty wash boat.

      2. Insert plate into base plate holder and remove plate cover.

      3. Replace pin block by guiding the rear dowels of pin block into the rear holes of the base plate.

      4. Push and hold the black lever.

      5. Lift pin block while continuing to hold the black lever down.

    3. Discard the medium using multi-channel pipette without disturbing the scratch.

    4. Add 200 μl of Low-serum culture medium (2% FBS) containing the experimental treatments.
      Note: We treat our synovial fibroblasts or human dermal fibroblasts with anti-citrullinated protein antibodies (ACPA) (Ossipova et al., 2014), control IgG, Tumor necrosis factor (TNF) and medium only.


  3. IncuCyte Zoom Scan setup (Figure 1A and Figure S1)

    To avoid interrupting an ongoing scan, it is very important to check device status. Place a new plate only between scheduled scans. Do not eject door during scanning (Figure S2).

    1. Place 96-well Imagelock plate and click ‘Schedule Scans’.

    2. Select the tray position of interest.

    3. Click ‘add vessel’ and select ‘96-well Essen Imagelock’ in Zoom software.

    4. Set scan type to ‘Scratch Wound’ with ‘Wide Mode’ and ‘Scan pattern’.

    5. Select ‘Phase contrast’ channels in Zoom software and Set plate layout as desired.

    6. Select the desired scan frequency and timing by, right clicking on the time base and selecting ‘Set Interval’. Scan interval depends on how many wells need to be scanned. We recommend a 1-2 h interval for a migration assay.

    7. Click ‘Apply’ button to finish setting. Any unapplied changes will not be performed.


  4. Plate Setup (optional) (Figure 1B)

    1. Under ‘properties’ panel, enter ‘label’, ‘cell type’, ‘passage’ and ‘notes’. In a high throughput assay, it is advisable to keep detailed information about experiments for further reference.

    2. Click ‘plate map’ and select ‘add’ in dropdown list to create assay layout. It is highly recommended to use plate map for further analysis.

    3. In plate map editor, click ‘NEW’ to create treatment.

    4. Select wells in the plate layout, enter concentration or dilution of treatment and add to selected wells.

    5. Apply step 4 to all treatments and complete plate map.

    6. Click ‘OK’ button to save plate map.



      Figure 1. An illustration of scan and plate setup for using IncuCyte Zoom system. Screenshots of the IncuCyte ZOOM software for Scan setup and Plate setup. A. Steps 1 to 6 show the procedure to setup plate position, scan mode, channel and intervals for scan setup. B. Steps 1 to 5 show the procedure to setup experiment properties, plate layout and treatment information.


  5. Data Collection and Processing

    1. In side menu, select the experiment that needs to be analyzed.

    2. Create or Add image collection (Figure 2A).

      1. In ‘analysis job utilities’, click ‘Create or Add image collection’.

      2. Select several images to create image collection for algorithm definition. It is recommended to select from different locations from plate layout and different time points from scan time to represent whole experiment.

      3. Save the image collection with appropriate title.

    3. New processing definition (Figure 2B).

      1. In ‘analysis job utilities’, click ‘New processing definition’.

      2. Select the newly created image collection and continue to define the scratching mask.

      3. In left side menu, drag slide bar towards either ‘background’ or ‘cell’ to adjust segmentation.

      4. In clean up panel section, enter a certain number to fill up the holes between cells (optional).

      5. In filter section, select ‘min’ in ‘Area’ and enter a number to exclude cell debris in the image.

      6. Select ‘phase contrast’ in Image Channel, ‘scratching wound mask’ and ‘confluence mask’ in Analysis Mask.

      7. Click ‘preview’ to check the current processing definition and click ‘preview all’ to apply current definition to all images in collection.

      8. Adjust the definition until it fits most of images in collection and save it with a proper name.

      9. The processing definition can be applied for batch experiments.

    4. Launch Analysis Job (Figure 2C).

      1. In ‘analysis job utilities’, click ‘Launch Analysis Job’.

      2. In pop-up window, enter the name of the analysis.

      3. Select start and finish time point to define time range.

      4. Select wells in plate layout and click ‘Launch’ to start analysis job.

      5. Check analysis result in main menu in ‘search’ and under ‘analysis job’ tab.


  6. Data Export (Figure 2D)

    1. In side menu, select the experiment that needs to be exported.

    2. Select ‘Metrics’ panel, choose appropriate ‘phase metrics’ and click ‘Graph/Export’ button.

    3. In pop-up window, select ‘wells’ and ‘time points/time range’ and click ‘Data Export’ button.

    4. In ‘Export Metrics’ window, select ‘Layout’, ‘Destination’, ‘Other option’ and Click ‘Export’ button to export data for further analysis.



      Figure 2. An illustration of data processing for using Cell Migration Analysis Software Module. Screenshots of the cell migration analysis software for data processing. A. Steps to select re-presentive images from all timepoints to create image collection. B. Steps to define confluence mask and wound mask for images from collection. C. Steps to launch analysis for selected plate and desired time range. D. Steps to export raw data.


  7. Lens Changing (Figure S3)

    IncuCyte Zoom offers three different optical (4×, 10×, and 20× lens). To perform migration assay, it is essential to have 10× lens. To minimize the risk of conflict, changing lens is only available via administer account.

    1. In main menu, go to ‘Task list’, click ‘administer’ and select tab ‘optical configuration’.

    2. Follow the steps of ‘optics configuration’ to install new lens.

    3. Accept the change and create new schedule.

Data analysis

  1. Representive example of data and confluence mask segmentation.

    When the analysis job is done, scratch wound mask, confluence mask and initial scratch wound mask are obtained at all timepoints. The wound confluence is simultaneously calculated by IncuCyte® Cell Migration Analysis Software. An example is shown in Figure 3: the confluence mask segmentation at the initial time point, end time point (Figure 3A) and wound confluence curve in time lapse, where cells were treated with antibodies: A, B and control antibody (Figure 3B).



    Figure 3. Example of confluence mask, scratch mask and analysis of wound confluence using IncuCyte Cell migration analysis software. Exported images from IncuCyte analysis software. A. A segmentation example of cell confluence mask (yellow) and scratch mask (red outline) at 0 h (initial time point) and 6 h (end point). B. An example of migration rate curve (wound confluence) between 0 to 24 h, where antibody A and B but not control antibody have effect on cell migration.


  2. Data analysis

    Incucyte Zoom software allows data export. The default figure of migration rate (percentage of wound confluence) is good enough for overview results. However, for statistical analysis, it is recommended to export raw data to Prism. Moreover, it is very useful to extract data and compare migration rates at specific time points. In Figure 4, we show migration rate, where fibroblasts were treated with ACPA, Ctrl IgG or medium only [The figure was originally published in Sun et al. (2019), Figure 1, as an open access article distributed in accordance with the Creative Commons Attribution 4.0 Unported (CC BY 4.0) license (https://creativecommons.org/licenses/by/4.0/)]. Data from same wells, with or without serum starvation, were analyzed for migration up to 20 h (Figures 4A and 4B) and images of the wound mask at time point 6 h is shown in Figure 4C. Data were normalized to the medium-treatment group at time point 6 h and presented as migration fold change (Figures 4D and 4E). We also performed cell migration assays on both human dermal fibroblasts (HDFs) and synovial fibroblasts of osteoarthritis patients (OASFs) with or without stimulation of IL-8 or TNF-alpha (Sun et al., 2019, Supplementary Figure 2).



    Figure 4. Increased mobility of synovial fibroblasts in the presence of polyclonal ACPAs. Real-time cell migration was measured in the presence of 1 µg/ml ACPA, control IgG or without any treatment in non-starved (A) and starved (B) fibroblast cultures using IncuCyte. Image-based evaluation of cell migration in starved fibroblast cultures were performed using Cell Migration Analysis software module with 10× magnification (C). Cell mobility was analyzed during a period of 6 h in the presence of 1 µg/ml polyclonal ACPA IgGs (ACPA) or non-ACPA control IgGs (IgG) or without antibody treatment (-) in both non-starved (D) and starved (E) fibroblast cultures. Dot line indicate migration index of non-treated fibroblasts. The graphs represent mean ± SD values of 6 replicates for each treatment. *P < 0.05.

Recipes

  1. Synovial fibroblasts culture medium (10% FBS)

    1. Dulbecco's Modified Eagle Medium (DMEM 500 ml)

    2. Add 50 ml heat-inactivated fetal bovine serum (FBS) to reach 10%

    3. Add 100 U/ml penicillin

    4. Add 100 µg/ml streptomycin

  2. Starvation medium (serum free)

    1. Dulbecco's Modified Eagle Medium (DMEM 500ml)

    2. Add 100 U/ml penicillin

    3. Add 100 µg/ml streptomycin

  3. Low-serum cell culture medium (2% FBS)

    1. Dulbecco's Modified Eagle Medium (DMEM 500ml)

    2. Add 10 ml heat-inactivated fetal bovine serum (FBS) to reach 2%

    3. Add 100 U/ml penicillin

    4. Add 100 µg/ml streptomycin

Acknowledgments

This project has received funding from FOREUM, Foundation for Research in Rheumatology, from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (grant agreement CoG 2017 - 7722209_PREVENT RA and grant agreement 777357_RTCure), from the Swedish Research Council and Konung Gustaf V:s och Drottning Victorias Frimurarestiftelse.

    Last, we would like to pay our gratitude and our respects to our group leader and colleague, Prof. Anca Catrina who passed away recently. She was a dedicated professor and excellent researcher in the Department of Medicine, Rheumatology Unit, at Karolinska University Hospital. We will continue her work and her style as much as we can.

    The protocol was first described in the methods section of Sun et al. (2019).

Competing interests

The authors declare that they have no conflicts of interest.

Ethics

This study involves human participants with ethical permit listed below:

1. Kartläggning av prediktiva biomarkörer vid kronisk artrit ID: 2009-358-31-3. (Mapping of predictive biomarkers in chronic arthritis).

2. Kartläggning av inflammatoriska mediatorers betydelse för sjukdomsförlopp vid kroniskaledsjukdomar ID:2009-1262-31-3. (Mapping of inflammatory mediators significance for disease course in chronic joint diseases).

References

  1. Krishnamurthy, A., Joshua, V., Haj Hensvold, A., Jin, T., Sun, M., Vivar, N., Ytterberg, A. J., Engstrom, M., Fernandes-Cerqueira, C., Amara, K., Magnusson, M., Wigerblad, G., Kato, J., Jimenez-Andrade, J. M., Tyson, K., Rapecki, S., Lundberg, K., Catrina, S. B., Jakobsson, P. J., Svensson, C., Malmstrom, V., Klareskog, L., Wahamaa, H. and Catrina, A. I. (2016). Identification of a novel chemokine-dependent molecular mechanism underlying rheumatoid arthritis-associated autoantibody-mediated bone loss. Ann Rheum Dis 75(4): 721-729.
  2. Liang, C. C., Park, A. Y. and Guan, J. L. (2007). In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro. Nat Protoc 2(2): 329-333.
  3. Ossipova, E., Cerqueira, C. F., Reed, E., Kharlamova, N., Israelsson, L., Holmdahl, R., Nandakumar, K. S., Engstrom, M., Harre, U., Schett, G., Catrina, A. I., Malmstrom, V., Sommarin, Y., Klareskog, L., Jakobsson, P. J. and Lundberg, K. (2014). Affinity purified anti-citrullinated protein/peptide antibodies target antigens expressed in the rheumatoid joint. Arthritis Res Ther 16(4): R167.
  4. Sun, M., Rethi, B., Krishnamurthy, A., Joshua, V., Circiumaru, A., Hensvold, A. H., Ossipova, E., Gronwall, C., Liu, Y., Engstrom, M., Catrina, S. B., Steen, J., Malmstrom, V., Klareskog, L., Svensson, C., Ospelt, C., Wahamaa, H. and Catrina, A. I. (2019). Anticitrullinated protein antibodies facilitate migration of synovial tissue-derived fibroblasts. Ann Rheum Dis 78(12): 1621-1631.

简介

[摘要]在此协议中,我们描述了一种通过贴壁细胞的活细胞成像监测细胞迁移的方法。刮擦测定法是研究细胞迁移或伤口愈合能力的常用方法。然而,实现均匀的scratc兴,发现为终点analys的最佳时间窗口我S和执行目标图像分析暗示,即使对于实施,并且熟练的实验者,对变异性和有限的再现性的高机会。因此,我们的协议通过使用均质伤口制作,顺序成像和自动图像分析来实现对细胞移动性的评估。细胞在96孔板中培养,和附着后,使用进行了由均质线状痕INCUCYTE ® W¯¯ oundMaker 。将处理直接添加到孔中,每2小时自动捕获一次图像。Ť此后,对图像进行的ProCE ssed通过定义刮擦掩模,并使用细胞汇合掩模软件算法。数据分析是进行使用的INCUCYTE ®细胞迁移分析软件。因此,我们的协议允许以高度可靠,可再现和可重新分析的方式对细胞迁移的治疗效果进行时滞分析。


[背景]划痕测定小号是用于研究细胞迁移一种广泛使用的方法或伤口愈合的能力。然而,常规方法(手动刮擦)需​​要技能来执行线性刮擦并且是终点测定(Liang等人,2007 ;Krishnamurthy等人,2016)。通常使用Ima geJ或其他软件手动分析数据。最近,我们在细胞迁移测定中采用了Essen Bioscience的高通量自动成像系统IncuCyte ZOOM (Sun等,2019)。通过使用INCUCYTE ® WoundMaker ,线状痕可均匀创建在最多至96孔在同一时间。用的适当的LY定义的算法,通过相衬分析,细胞汇合掩模小号和刮伤掩模小号,细胞迁移,可同时评估。简言之,该常规方法是比较费力且耗时比我们在座的方法。该协议为处理高通量样本和随时间推移以无偏方式分析数据提供了时间和精力最少的方法。

关键字:细胞迁移, 划痕法, 活细胞成像, 延时成像, 高通量

材料和试剂

1. INCUCYTE ® ImageLock 96孔板(Essen的BIOSCI ENCE,目录号:4379)     

2.滑膜成纤维细胞(从接受关节置换的类风湿性关节炎患者中分离,Sun等人,2019年)     

3.正常人皮肤成纤维细胞(PromoCell ,目录号:C-12300)     

4.原发性人类骨关节炎滑膜成纤维细胞(Bioivit ,目录号:HPCSFOA-03)     

5. Dulbecco改良的Eagle培养基(DMEM)(Sigma-Aldrich,目录号:D5796-500ml)     

6.胎牛血清(FBS)(Sigma-Aldrich,目录号:F7524)     

7.胰蛋白酶-EDTA(Sigma-Aldrich,目录号:T3924-100ml)     

8.磷酸盐缓冲盐水(PBS)(Sigma-Aldrich,目录号:D8537-500ml)     

9.青霉素链霉素(PEST)(Sigma-Aldrich,目录号:P4333-100ml)     

10.抗瓜氨酸化蛋白抗体(从RA患者的外周血中纯化,Ossipova等人,2014 ) 

11.重组人TNF-α(Peprotech ,目录号:300-01A) 

12.重组人IL-8 / CXCL8蛋白(R&D Systems,目录号:208-IL-010) 

13. Alconox粉末(VWR,目录号:21835-123) 

14.香囊,依靠+论TM卫可®粉末(VWR,目录号:148-0200) 

15.无菌蒸馏水(室内生产) 

16. 70%乙醇(Sigma-Aldrich,目录号:470198-1L) 

17.滑膜成纤维细胞培养基(10%FBS)(请参阅食谱) 

18.饥饿培养基(无血清)(请参阅食谱) 

19.低血清细胞培养基(2%FBS)(请参阅食谱) 



设备

INCUCYTE ® WoundMak呃有两个洗船(埃森生物科学,目录号:4493 )
INCUCYTE ZOOM活细胞分析系统(埃森生物科学,型号:INCUCYTE ® ZOOM)
多通道移液器,8通道,20-200 μ升(VWR,符合人体工学的高性能多通道移液器,目录号:89079-948)               


软件

细胞迁移分析软件模块(Essen Bioscience,目录号:4400)
Prism 6(Gra phPad软件)
INCUCYTE ®变焦软件(2018A)


程序

准备细胞
种子细胞在150 μ升完全生长培养基中96孔imagelock板使用多通道移液管在细胞密度,将达到高达95%汇合过夜。在96孔板中,由于蒸发作用,建议将外部孔排除在实验之外。
注意:我们每孔播种20,000个滑膜成纤维细胞或人真皮成纤维细胞,以在24小时内完全融合。


填充外,我们用300 LLS微升PBS在井的其余部分抵消蒸发效应。
在加有5%CO 2的潮湿培养箱中于37 °C过夜培养细胞,直到细胞达到95%融合为止。
洗涤细胞用100微升PBS洗涤两次使用一个多通道移液器。
Starv E细胞瓦特第i 100微升FBS不含培养基在加湿培养箱用5%CO 2 2小时以耗尽生长因子。
注意:在我们的环境中,我们使细胞饥饿2小时。隔夜饥饿通常用于生长因子的消耗。饥饿时间可能不同,是否取决于荷兰国际集团上的实验人设置。

划伤
清洁伤口机的洗涤船,每次5分钟在一连串四个洗涤溶液(45毫升每个)
0.5%Alconox
1%维康
无菌蒸馏水
70%乙醇
使用该INCUCYTE ®伤口设备制造同质划痕
(https://www.youtube.com/watch?v=x7pMzJ1VIdA&feature=youtu.be)


取下创口机的顶部,然后将其放在空的清洗船中。
将板插入底板支架中,然后取下板盖。
将插销块的后销钉插入基板的后孔中,以更换插销块。
按住黑色手柄。
继续按住黑色手柄的同时提起销钉块。
使用多通道移液器丢弃培养基,而不会干扰刮擦。
加入200μl含实验处理剂的低血清培养基(2%FBS)。注意:我们仅使用抗瓜氨酸化蛋白抗体(ACPA)(Ossipova et al。,2014),对照IgG,肿瘤坏死因子(TNF)和培养基来治疗滑膜成纤维细胞或人真皮成纤维细胞。


INCUCYTE缩放扫描设置(图1A和˚F igure š 1)
为了避免中断了正在进行的扫描,这是非常重要的检查德维ç E的身份。P蕾丝一个新盘只计划扫描之间。扫描时切勿退出门(˚F igure š 2)。


放置96孔I Magelock板,然后单击“计划扫描” 。
选择所需的纸盘位置。
单击“添加容器”,然后在Zoom软件中选择“ 96孔Essen Imagelock ” 。
使用“宽模式”和“扫描模式”将扫描类型设置为“划伤伤口”。
在Zoom软件中选择“相位对比”通道,然后根据需要设置板布局。
通过右键单击时基并选择“设置间隔”来选择所需的扫描频率和定时。扫描间隔取决于需要扫描的孔数。我们建议一个1-2小时,间隔一个迁移试验。
点击“应用”按钮完成设置。任何未应用的更改将不会执行。


印版设置(可选)(图1B)
在“属性”面板下,输入“标签” ,“单元格类型”,“段落”和“注释”。以高通量测定法中,可取的是保持详细编信息有关实验用于进一步参考。
单击“板图”,然后在下拉列表中选择“添加”以创建化验布局。强烈建议使用板图进行进一步分析。
在板图编辑器中,单击“新建”以创建处理。
在板布局中选择孔,输入处理的浓度或稀释度,然后添加到选定的孔中。
将步骤4应用于所有处理并完成板图。
单击“确定”按钮保存板图。




图1.使用IncuCyte Zoom系统进行扫描和印版设置的图示。用于扫描设置和印版设置的IncuCyte ZOOM软件的屏幕截图。一。步骤1至6显示了设置印版位置,扫描模式,通道和扫描间隔的步骤。B.步骤1至5显示了设置实验属性,板布局和处理信息的过程。


数据收集和参考程序ssing
在侧面菜单中,选择的实验,需要小号进行分析。
创建或添加图像集合(图2A)。
在“分析作业实用程序”中,单击“创建或添加图像集合”。
选择多个图像以创建图像集合以进行算法定义。建议从板的布局中选择不同的位置,并从扫描时间中选择不同的时间点以代表整个实验。
保存具有适当标题的图像集。
新的处理定义(图2B)。
在“分析作业实用程序”中,单击“新处理定义”。
选择新创建的图像采集,并继续定义的划伤面罩。
在左侧菜单中,将滑动条拖向“背景”或“单元格”以调整分段。
在清理面板部分中,输入一定的数字以填充单元格之间的孔(可选)。
在“过滤器”部分中,在“区域”中选择“最小”,然后输入数字以排除图像中的细胞碎片。
在“图像通道”中选择“相衬”,在“分析蒙版”中选择“抓伤伤口面膜”和“融合面膜”。
单击“预览”以检查当前处理定义,然后单击“预览全部”以将当前定义应用于集合中的所有图像。
调整清晰度,直到它适合收藏中的大多数图像为止,并以适当的名称保存。
该凝固酶原ssing定义可应用于批量实验。
启动分析作业(图2C)。
在“分析作业实用程序”中,单击“启动分析作业”。
在弹出的窗口中输入名称的分析。
选择开始和结束时间点以定义时间范围。
在板布局中选择孔,然后单击“启动”以开始分析作业。
在“搜索”和“分析工作”选项卡下的主菜单中检查分析结果。


数据导出(图2D)
在侧面菜单中,选择的实验,需要小号要出口。
选择“指标”面板,选择适当的“阶段指标”,然后单击“图形/导出”按钮。
在弹出窗口中,选择“孔”和“时间点/时间范围”,然后单击“数据导出”按钮。
在“导出指标”窗口中,选择“布局”,“目标”,“其他选项”,然后单击“导出”按钮以导出数据以进行进一步分析。




图2.数据的图示的ProCE ssing用于使用细胞迁移分析软件模块。对数据的细胞迁移分析软件的屏幕截图凝固酶原ssing 。A.步骤选择重新presentive从所有图像的时间点来创建图像采集。B.为收集的图像定义融合口罩和伤口口罩的步骤。C.启动针对选定板块和所需时间范围的分析的步骤。D.导出原始数据的步骤。

透镜起了变化克(˚F igure š 3)
INCUCYTE变焦报价小号三个不同的光(4 × ,10 ×和20 ×透镜)。要进行迁移测定,必不可少的是10倍透镜。为了最大程度地减少发生冲突的风险,只能通过管理帐户来更换镜头。


在主菜单中,转到“任务列表”,单击“管理”,然后选择选项卡“光学配置” 。
按照“光学配置”中的步骤安装新镜头。
接受更改并创建新时间表。


数据分析

数据和汇合蒙版分割的代表性示例。
当分析工作的完成,划痕面具,面具汇合和初始划痕面膜是在所有获得的时间点。伤口合流是S imultaneously通过计算INCUCYTE ®细胞迁移分析软件。一个例子示于图3 :合流掩模分割在所述初始时间点,结束时间点(图3A)和伤口合流曲线在时间流逝,其中细胞用抗体处理:A,B和对照抗体(图3B) 。



图3.融合面罩,防刮面罩和使用IncuCyte Cell迁移分析软件分析伤口汇合的示例。从IncuCyte分析软件导出的图像。A.在0小时(初始时间点)和6小时(终点)时,细胞融合口罩(黄色)和刮擦口罩(红色轮廓)的分割示例。B. 0至24小时之间迁移率曲线(伤口融合)的例子,其中抗体A和B但对对照抗体没有影响,对细胞迁移有影响。

数据分析
Incucyte Zoom软件允许数据导出。默认的迁移率数字(伤口汇合百分比)足以获得概览结果。但是,为了进行统计分析,建议将原始数据导出到Prism。此外,提取数据并比较特定时间点的迁移率s非常有用。在图4中,我们显示迁移率,其中成纤维细胞进行处理以ACPA,Ctrl键IgG或仅培养基[该图最初发表于孙等人。(2019 ),图1中,作为分布式按照CC署名4.0 Unported(CC BY 4.0)许可开放接入文章(https://creativecommons.org/licenses/by/4.0/)] 。数据从同一孔中,有或没有血清饥饿,分析了迁移到20小时(图4A和4B)和图像的在时间点6伤口掩模ħ被示于图4C。数据归一化到所述介质处理组在时间点6小时,并呈现为迁移的倍数变化(图4D和4E)。我们还进行细胞迁移测定小号上人类真皮成纤维细胞(HDF)中的和骨关节炎的患者(OASFs)具有或不具有IL-8或TNF-α刺激的滑膜成纤维细胞(孙等人,2019,补充图2) 。



图4.在存在多克隆ACPA的情况下滑膜成纤维细胞的活动性增加。使用IncuCyte,在非饥饿(A)和饥饿(B)成纤维细胞培养物中,在存在1 µg / ml ACPA,对照IgG或不进行任何处理的情况下,测量实时细胞迁移。使用细胞迁移分析软件模块以10倍放大倍数(C)对饥饿的成纤维细胞培养物中的细胞迁移进行基于图像的评估。在无饥饿(D)和饥饿的情况下,在1 µg / ml多克隆ACPA IgG (ACPA)或非ACPA对照IgG (IgG)或未经抗体处理(-)的情况下,在6小时内分析细胞迁移率(五)成纤维细胞培养。点线表示未处理的成纤维细胞的迁移指数。这些图表示平均值± SD值的6个重复每个处理。* P < 0.05






菜谱

滑膜成纤维细胞培养基(10%FBS)
Dulbecco的改良Eagle培养基(DMEM 500毫升)
加入50 ml热灭活胎牛血清(FBS)达到10%
加入100 U / ml青霉素
加入100 µg / ml链霉素
饥饿培养基(无血清)
Dulbecco的改良Eagle培养基(DMEM 500毫升)
加入100 U / ml青霉素
加入100 µg / ml链霉素
低血清细胞培养基(2%FBS)
Dulbecco的改良Eagle培养基(DMEM 500毫升)
加入10 ml热灭活胎牛血清(FBS)达到2%
加入100 U / ml青霉素
加入100 µg / ml链霉素


致谢

该项目已获得瑞典研究机构FOREUM,风湿病研究基金会和欧洲研究理事会(ERC)资助的欧盟Horizon 2020研究与创新计划(CoG 2017-7722209_PREVENT RA和拨款协议777357_RTCure)理事会和Konung古斯塔夫五:S ^ OCH Drottning维多利亚Frimurarestiftelse 。


最后,我们要感谢我们的小组负责人和同事安卡·卡特里娜(Anca Catrina)教授,他最近去世了。她是卡罗林斯卡大学医院风湿病科医学系的一名专门教授和优秀研究员。我们将尽可能地继续她的工作和她的风格。


该协议首先在Sun等人的方法部分中进行了描述。(2019 )。

利益争夺

作者宣称他们没有利益冲突。

伦理

这项研究涉及具有以下道德许可的人类参与者:


1. KartläggningAV prediktivabiomarkörerVID kronisk artrit ID:2009-358-31-3 。(在慢性关节炎预测性生物标记的映射)。           

2.炎症介质的调解员,编号:2009-1262-31-3。(炎症介质意义作图病程在慢性关节疾病)。           



参考

Krishnamurthy,A.,Joshua,V.,Haj Hensvold ,A.,Jin,T.,Sun,M.,Vivar ,N.,Ytterberg ,AJ,Engstrom ,M.,Fernandes-Cerqueira ,C.,Amara,K 。,Magnusson,M.,Wigerblad ,G.,Kato,J.,Jimenez-Andrade,JM,Tyson,K.,Rapecki ,S.,Lundberg,K.,Catrina,SB,Jakobsson ,PJ,Svensson ,C. ,Malmstrom ,V.,Klareskog ,L.,Wahamaa ,H.和Catrina,AI(2016)。类风湿关节炎相关的自身抗体介导的骨丢失基础的新型趋化因子依赖的分子机制的鉴定。Ann Rheum Dis 75(4):721-729。
Liang,CC,Park,AY和Guan,JL(2007)。体外划痕测定:一种方便且廉价的体外细胞迁移分析方法。纳特Protoc 2(2):329-333。
Ossipova,E.,Cerqueira,CF,Reed,E.,Kharlamova,N.,Israelsson,L.,Holmdahl,R.,Nandakumar,KS,Engstrom,M.,Harre,U.,Schett,G.,Catrina, AI,Malmstrom,V.,Sommarin,Y.,Klareskog,L.,Jakobsson,PJ和Lundberg,K.(2014)。亲和纯化的抗瓜氨酸化蛋白/肽抗体靶向类风湿关节中表达的抗原。Arthritis Res Ther 16(4):R167。
Sun,M.,Rethi,B.,Krishnamurthy,A.,Joshua,V.,Circiumaru,A.,Hensvold,AH,Ossipova,E.,Gronwall,C.,Liu,Y.,Engstrom,M.,Catrina ,SB,Steen,J.,Malmstrom,V.,Klareskog,L.,Svensson,C.,Ospelt,C.,Wahamaa,H. and Catrina,AI(2019)。抗瓜氨酸化蛋白抗体有助于滑膜组织来源的成纤维细胞的迁移。Ann Rheum Dis 78(12):1621-1631。
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引用: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.
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