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Nov 2018

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Automated Analysis of Cell Surface Ruffling: Ruffle Quantification Macro
细胞表面Ruffling的自动分析:Ruffle的宏观量化   

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Abstract

Cell surface protrusions include F-actin rich, wave-like ruffles that are erected transiently in response to stimuli and during cell migration. Macrophages are innate immune cells that ruffle constitutively and more dramatically in cells activated by pathogens. Dorsal ruffles and their resulting macropinosomes are key sites for environmental sampling, pathogen detection and immune signaling. Quantitative assessment of ruffling is important for assessing pathogen responses in macrophages and for analysis of growth factor responses in other cell types but automated and quantitative methods are lacking, and rely on manual and qualitative assessments. Here we present an automated ImageJ macro for quantifying dorsal cell surface protrusions from 3D microscope images. The assay presented here is suitable for high-throughput screening applications to detect drug, pathogen, or growth factor induced changes in cell ruffling by measuring ruffle area and intensity and providing normalized values in an easy to read combined spreadsheet.

Keywords: Ruffling (Ruffling), Image analysis (图像分析), F-actin (F-肌动蛋白), Cell surface (细胞表面), ImageJ Macro (ImageJ Macro), Automation (自动化), High-throuhgput (高通量), Microscopy (显微镜)

Background

Cells develop ruffles or erect veils of membrane on their surfaces during migration and in responses to growth factors or other mediators. As innate immune cells, macrophages ruffle both constitutively and upon stimulation by growth factors, pathogens or other activating stimuli. Ruffles can take on different forms. Dorsal ruffles on the upper surface of adherent cells are typically large erect. F-actin-rich veils which can be circular (hence the alternative name ‘circular dorsal ruffles’) usually rise up transiently and then collapse back onto the surface. Ruffles often form macropinosomes as described in Condon et al., 2018. Macropinosomes internalise the ruffle membrane, simultaneously ingesting fluid and soluble or small particulate cargo (Kerr and Teasdale, 2009; Commisso et al., 2013). Dorsal ruffles or circular dorsal ruffles have been described in depth in macrophages, epithelial cells and fibroblasts where they have gained recognition as sites for immune cell activation (Luo et al., 2014; Wall et al., 2017), growth factor signaling (Buccione et al., 2004) and activated receptor clustering, and internalisation that play pivotal roles in cancer cell signaling (Buccione et al., 2004; Orth and McNiven, 2006). Dorsal ruffles are also induced by a number of pathogens, including Salmonella entericia serovar typhimurium at sites of pathogen entry (Ly and Casanova, 2007).

The large, circular dorsal ruffles that have been observed on different cell types are dependent on the transient production of F-actin, and inhibitors of F-actin polymerisation such as cytochalasin D abolish their formation (Hedberg et al., 1993; Buccione et al., 2004). As dorsal ruffles are highly enriched in F-actin and contribute a much greater area than other F-actin rich projections, such as filopodia, on the dorsal surface, fluorescent markers of F-actin are relatively enriched in dorsal ruffles. Many studies utilize the presence of dorsal ruffles as a qualitative read-out of cellular responses (Gurung et al., 2003; Gandhi et al., 2004; Singla et al., 2019). The ability to quantify ruffling or F-actin cell surface protrusions, would provide useful analysis for a wide range of biological research ranging from stimulation assays to high-throughput screening. Ruffling has been assessed in images by taking whole-cell, whole field of view F-actin measurements (Schlam et al., 2016) or by performing manual selection and user-interactive regions of interest (Venter et al., 2015).

Here, we have developed a robust and automated assay for dorsal ruffle quantification that requires no user input for cell selection or segmentation of both basal and dorsal regions, defined by the mid-point of the nucleus. Measurements for ruffles are also made on Sum-slices images, meaning depth information is taken into account (i.e., taller ruffles contribute to higher intensity counts). This assay enables batch processing of multiple images to analyse large cell numbers and provide tabulated results for easy statistical analysis using third party software. This robust, automated assay is well suited for screening cell populations to detect changes in dorsal ruffling induced by extracellular stimuli such as pathogens, growth factors, hormones or cytokines and/or large scale screening of pharmacological inhibitors, natural products, or nutritional conditions.

Materials and Reagents

  1. Polystyrene Petri Dish for cell passaging (Thermo Fisher Scientific, catalog number: LBSPD1002X, or equivalent)
  2. 24-well tissue culture plate (Thermo Fisher Scientific, catalog number: 142475, or equivalent)
  3. 12 mm round #1 coverslip (Thermo Fisher Scientific, catalog number: MENCSC121GP, or equivalent)
  4. Microscope slide 26 x 76 mm (VWR, catalog number: VWRI631-1558, or equivalent)
  5. RAW264.7 macrophage-like cell line (ATCC, catalog number: TIB071)
  6. Clear Nail Polish (Ultra3, catalog number: 14111130, or equivalent)
  7. RPMI 1640 media (Lonza Australia, catalog number: BE12-702F)
  8. Fetal Bovine Serum (Thermo Fisher Scientific, catalog number: 16000044)
  9. L-glutamine 100x (Thermo Fisher Scientific, catalog number: 25030-081)
  10. 4% Paraformaldehyde (ProSciTech, catalog number: EMS15735-100, or equivalent)
  11. Triton X-100 (Sigma-Aldrich, catalog number: X100-500ML)
  12. Phosphate buffered saline (PBS) (Thermo Fisher Scientific, catalog number: 10010023, or equivalent)
  13. AlexaFluor 488-Phalloidin (Invitrogen, catalog number: A12379, see Notes)
  14. DAPI nuclei stain (Roche, catalog number: 10236276001, or equivalent, see Notes)
  15. ProLong Diamond Antifade reagent (Thermo Fisher Scientific, catalog number: P10144, or equivalent)
  16. Poly-D-Lysine (Thermo Fisher Scientific, catalog number: A3890401, or equivalent)
  17. Cell culture medium (see Recipes)

General

  1. Cell passaging supplies
  2. 15 ml conical tubes
  3. Pipette tips (10, 50, 100, 1,000 μl)
  4. Kimwipes
  5. Lens wipes
  6. Parafilm
  7. Paper towel
  8. Immersion oil
  9. dH2O
  10. 70% Ethanol (EtOH)

Equipment

  1. Automated serological pipette (Thermo Fisher Scientific, S1 Pipette Filler, catalog number: 14-387-163, or equivalent)
  2. Graduated pipettes (P2, P20, P100, P1000) (John Moris, Gilson #115209, or equivalent)
  3. Precision forceps (Dumont, style 7, catalog number: T07-912, or equivalent)
  4. Temperature and CO2 controlled incubator (VWR, catalog number: 390-2012, or equivalent)
  5. Tissue culture inverted phase contrast microscope
  6. Hemocytometer
  7. Light-proof staining chamber
    Note: An aluminum foil coated shallow chamber is sufficient.
  8. Zeiss AxioImager Upright fluorescence Apotome Microscope (see Notes, or equivalent)

Software

  1. Zeiss Zen Blue 2.3 (Carl Zeiss, Germany, or equivalent acquisition software)
  2. FIJI Image Analysis program (Schindelin et al., 2012)
  3. Ruffle Quantification Macro (https://github.com/NickCondon/RuffleQuantification)
  4. Microsoft Excel (or equivalent)
  5. GraphPad Prism 8 (or equivalent)

Procedure

  1. Cell culture
    1. Create a suspension of your cell line choice.
    2. Determine cell concentration using hemocytometer and dilute to a concentration 0.1 x 106 cells/ml in tissue culture medium (Recipe 1).
    3. Place one coverslip into each well of 24-well tissue plate.
    4. Add 500 μl of diluted cell suspension (0.5 x 105 cells) to each well.
      Note: Desired confluency the next day = 60%.
    5. Press coverslip into base of well using forceps to remove any air bubbles that have formed under the coverslip.
    6. Allow cells to adhere and spread out overnight (at least 16 h) prior to experiments.
      Note: For longer treatment or transient transfection, lowering the initial cell seeding density may be required (60% confluency on day of fixation is desirable).
    7. Perform stimulation/pharmacological inhibition experiment as required.
      Note: We routinely use 10-100 ng/μl lippopolysaccharide for 30 min to induce ruffle formation.
    8. Aspirate media and wash with ice cold PBS.
    9. Fix cells for at least 30 min with 4% Paraformaldehyde.
    10. Thrice wash cells with room temperature PBS.

  2. Staining coverslips
    1. Place a wet paper towel into the base of the staining chamber.
    2. Lay out parafilm on top of wet paper towel.
    3. Remove coverslips from 24-well plate and place cell-side up onto parafilm.
    4. Add a drop of PBS onto each coverslip.
      Note: Coverslips should be kept ‘wet’ at all times to ensure cell integrity and to prevent dehydration.
    5. Permeabilise cells by placing a drop of 0.1% Triton X-100 onto each coverslip for no more than 5 min.
    6. Wash coverslips with PBS three times by aspiration.
    7. Prepare AlexaFluor 488-Phalloidin and DAPI (2 mg/ml) labelling mix by diluting stock solutions 1:500, and 1:1,000 respectively into ice cold PBS.
    8. Place a 50 μl drop of labelling mix onto parafilm adjacent to each coverslip.
    9. Aspirate PBS from coverslip and invert the coverslip onto the drop of labelling mixture (cell side down).
    10. Incubate for at least 30 min at room temperature (RT, 25 °C), protected from the light.
    11. Invert coverslips and wash with PBS three times.
    12. Wash coverslips with dH2O once to prevent PBS crystals forming.
    13. Place a small drop of mounting medium (we use ProLong Diamond) onto microscope slides .
      Note: For best results microscope slides should be wiped with 70% EtOH prior to use to remove any dust and lint.
    14. Lift coverslip from parafilm using forceps and remove any excess water by gently placing the edge of the coverslip against a clean Kimwipe tissue.
    15. Place the coverslip cell side-down onto the drop of mounting medium.
    16. Gently press the forceps against the top of the coverslip and aspirate any excess mounting medium.
    17. Seal the coverslip to the microscope slide using clear nail polish.

  3. Imaging and Data Analysis
    1. Capture 3D z-stacks using Nyquist sampling with a fluorescent microscope capable of serial optical sections, i.e., Apotome, Confocal or widefield system (Figures 1A-1C) to capture the full 3-dimensional image of the cells and ruffles.
    2. Acquire multiple fields of view for each condition (collecting at least 100 cells per treatment).
      Notes:
      1. Images here were captured using a Zeiss Axioscan fluorescence upright microscope using a 40x 1.3 NA Plan Neofluar objective with an Axiocam 506 CCD camera. Samples were illuminated with HXP 120W whitelight source and filtered with Zeiss DAPI 49/FITC 44 filter cubes. Z-stacks were captured at 0.2 μm intervals beyond both the visible top and bottom of the cells with 5 apotome phase shifts for optical sectioning to identify the midpoint of the nucleus and ruffles on the dorsal surface (Figures 1D-1F).
      2. Nuclei Mid-point Offset determination. In its native form the script determines the mid-point of the nucleus by finding the slice where the cross-sectional area is greatest (Figure 1G). In macrophages, this suitably allows for the selection and segmentation of dorsal ruffles which are only present above this mid-point, however for alternate use cases, or different cell lines it may be required to use a different selection cut-off point. Figure 1H shows where peripheral ruffles may not be originally selected by using the nuclei mid-point, instead by applying an offset (of a few slices) the additional peripheral ruffle is selected (Figure 1H inset, grey arrow). The nuclei mid-point offset value allows for greater flexibility and broadens the use of the script to further analysis applications.


      Figure 1. Processing steps of the Ruffle Quantification Macro. 3-Dimensional, 2 channel images are required for the macro captured here using a 40x 1.3 NA objective. Images A-C represent Z-projected (for graphical display purposes only) example images. A. Phalloidin stained RAW 264.7 Macrophages (channel 1). B. Nuclei labelled with DAPI (channel 2). C. Merged two channel image, solid arrow represents location of reslice. D-F. Reslices of C (arrow), showing X-Z axis (cut vertically along the arrow in panel C) of 3D images. D. Dotted line represents mid-point of the nucleus, as selected by the macro E-F. F-actin rich ruffles with merged DAPI highlighted by white arrows (F). Scale bar = 20 μm. G. Example cross-sectional images (X-Y axis) from three different Z-planes of the nuclei showing differences in the measured area, with the largest cross-sectional area determined to be the nuclei ‘mid-point.’ H. Example resliced (X-Z axis) of cell showing a solid line where the determined mid-point of the nucleus is, as well as where the offset line would be if used (example, -3 offset), small open arrows (grey) highlight additional ruffle that would be selected if offset is used, larger black arrows show ruffles selected by the existing mid-point slice with the differential between slices being the offset.

    3. Utilising FIJI, install the macro file: ‘Ruffle Analysis Macro’ and select Run.
      Note: To load the macro into FIJI either open the .ijm file, or copy the text from the Github website (see Software section above) and paste this into a new script window (File>New>Script). For continued use, the file Ruffle_Analysis_Macro_.ijm can be copied into the “scripts” folder of the FIJI application, where it will appear under the plugins menu from the next time you open FIJI.
    4. Read the Acknowledgement screen for assay description and instructions (Figure 2A).
    5. Navigate to the directory location for your files for processing (Figure 2B).
    6. Confirm the file extension in use (e.g., “.czi”) in the dialog box and enter a Nuclei Mid-Point Offset if required and select “OK” (Figure 2C).
      Note: The script will run through the entire directory only opening images that contain the file extension chosen.
      Upon completion of the script a dialog will appear.
    7. Confirm success running of the macro with the batch completion dialog box (Figure 2D).
    8. Navigate to the output directory location (within the chosen working directory location) and confirm thresholding selections are accurately selecting your regions of interest using the output threshold images.
      Note: Due to the streamlined nature of the macro, the time required to re-run the macro with new or modified parameters is minimal and it is encouraged that output data is checked and re-run if necessary.


      Figure 2. User-interactive macro windows. Dialog boxes requiring user-input during the running of the Ruffle Quantification Macro. A. Acknowledgement and splash screen describing the macro, contact details of the creator, input requirements, and output files. B. OS-dependent file navigator window to select the directory containing images for processing and analysis. C. File extension filter dialog window allows for the user to enter the extension for file filtering and mid-point offset value. D. Macro completion window to notify the user the successful completion of the macro.

  4. Overview of key steps for Ruffle Quantification Macro
    1. Nuclei images are extracted (channel 2) and thresholded using the Otsu algorithm. The mid-point is determined by progressively measuring nuclei area within each slice. The slice with the greatest area of the nuclei is stored. The number of nuclei is also counted and stored for output (Figure 3A).
      Note: The Otsu thresholding method is used here to detect nuclei, the Otsu method finds the maximum point of inter-class variance between the foreground and the background and is suited where bi-modal distributions of intensity are expected.
    2. The F-actin (phalloidin) channel (channel 1) is extracted from the image stack into two separate images stacks, (1) the bottom half of the cell is taken (slice 1 to nuclei midpoint slice ( offset); Figure 3B) and (2) the top, ruffle area is taken (midpoint slice slice ( offset) to the top most slice; Figure 3E).
    3. The cell area is determined by maximum Z-projecting the image slices (from slice 0 to the determined nuclei midpoint slice (± offset) and thresholding using the Triangle algorithm (to select total cell peripheries). and measured for output (Figure 3C). The thresholded flattened bottom image is then reduced using a distance map to separate close and/or touching cells. The distance map is then thresholded using the IJ_IsoData threshold algorithm and the number of ‘objects’ (cells) is estimated and stored for output (Figure 3D).
      Note: The Triangle algorithm is used here to detect cells, the algorithm is best suited for finding objects that may only be slightly above background (e.g., the very edges of cells) and is therefore used to create accurate selections for measuring cell area. For cell counting, the distance mapped thresholded cells are subjected to a Moments IJ_IsoData threshold as it selects for high-value maximums (e.g., the very center or cells) and thus will not be influenced by touching cells, provided accurate cell number counts.
    4. The ruffles are determined by sum Z-projecting the top F-actin channel (midpoint ± offset to total/top number of slices) and measuring the intensity of the resultant 32-bit image (multiplied by the detected area). The flattened top/ruffle image is then thresholded using the Moments algorithm and the area of the thresholded ruffles is measured (Figure 3F) and used to determine regions of interest for directly measuring summed ruffle intensity.
      Note: The Moments algorithm is used here to threshold the ruffles, the algorithm works on a series of averaging steps, and selects regions above background but of varying intensity. This facilitates an accurate selection of all ruffles from the 32-bit sum projected image and not just the brightest objects.
    5. Calculations perform normalisations of ruffle area/cell area, and the intensity of the ruffles/cell area. These values are further normalized against the number of nuclei detected.
    6. Images at key points in the macro are saved into the output folder (nuclei detection, cell area (bottom) and ruffle area (top), and a merged (green/red) RGB image of the ruffle selection overlayed the cell area image) for verification purposes (Figures 3G-3H).
    7. All output values are written to the log window (Figure 3I), and the output .csv file into a subdirectory called Analysis_Results_ (Figure 3J).
    8. Quantified data from the analysis is stored as a .csv file with ratios and normalization to cell number already calculated (Figure 3K). (Values and calculations explained in detail below).


      Figure 3. Processing steps of the Ruffle Quantification Macro. A. Thresholded nuclei mask as counted by the macro. B-C. Flattened F-actin base of the cell (bottom) and corresponding threshold for measuring cell area. D. Thresholded distance map showing individual cell segmentation for counting number of cells/field of view. E-F. Sum Z-projected top surface (ruffles) including thresholded image for measuring ruffle area. G. Example output image showing merged bottom (red) and top/ruffles (green). H. Example output image showing merged thresholded bottom (red) and top/ruffles (green) I. Screenshot of the Log window reporting status, macro run date, file names and results. J. Screenshot of the results directory containing output images, log file and a Results.csv file. K. Screenshot of the .csv output results file with explanation of source information below referring to images above where applicable. Scale bar = 20 μm.

  5. Overview data processing steps
    1. Output data (.csv file) from the macro is opened using Microsoft Excel (or equivalent).
    2. Data where the nucleus mid-point is incorrect (e.g., slice 33/33) suggests incorrect thresholding and detection these samples are excluded from the analysis, this can be further confirmed from the corresponding threshold output images.
    3. Data are copied and sorted into relevant groups (based upon image fields of view and/or treatment conditions) into GraphPad Prism 8 for statistical analysis.
    4. Depending on the data-sets. statistical analysis may include T-test, One- or Two-way ANOVAs. Data are compared using the in-built graphing tools.

  6. Output and Results of the Macro
    The following details the source of values generated within the output .csv file:
    Column A (Image ID) = Filename of source image for this row of data
    Column B (Est. Number of cells) = Count of objects detected in Figure 3D
    Column C (Number of Nuclei) = Count of objects detected in Figure 3A
    Column D (Middle Slice#) = Image slice number of mid-point of nuclei
    Column E (Total Slice#) = Number of slices in the source Z-stack
    Column F (Total Cell Area) = Calculated area from Figure 3C
    Column G (Sum Intensity) = Sum intensity from Figure 3E
    Column H (Top Area Ruffles) = Calculated area from Figure 3F
    Column I (Ratio Top:Bottom) = Ratio Area from Figure 3F/ Area Figure 3C
    Column J (Ratio Sum Intensity:Cell Area) = Ratio Sum intensity Figure 3E/ Area Figure 3C
    Column K (Normalised Ratio) = Result of Column I (Figure 3F/3C) divided by Column C (Number of Nuclei Figure 3A)
    Column L (Normalised Sum) = Result of Column J (Figure 3E/3C) divided by Column C (Number of Nuclei Figure 3A)

Notes

  1. For these experiments the Zeiss AxioScan microscope with Apotome 2. was used to generate fluorescent images, however any fluorescence microscope with optical sectioning ability (e.g., confocal) can be substituted. Importantly, images must be 3-dimensional with the full range of the cells that are imaged, (from below the base, to above the top-most ruffle).
  2. Here, F-actin was labelled with Alexafluor 488-phalloidin, however any colour phalloidin can be substituted. Additionally, this macro works with genetically encoded F-actin labels such as GFP-LifeAct. Nuclei were stained with DAPI, however other nucleus/DNA markers would likely be suitable as well for determining the mid-point of the nuclei as part of the script.
  3. For these experiments RAW264.7 macrophage cells were utilized, however this assay has also been tested on primary bone-marrow derived macrophages (BMMs), MB231 cancer cells and BV2 microglial cells. The Ruffle Quantification Macro is therefore well suited to be used on multiple different cell types, whereby specific cell culture techniques may be modified.

Recipes

  1. Cell culture medium
    RPMI 1640 Culture Medium
    10% Feotal Bovine Serum
    1% Poly-D-Lysine

Acknowledgments

Microscopy was performed at IMB Microscopy and the Australian Cancer Research Foundation (ACRF) funded Cancer Biology Imaging Facility at IMB. NDC received support from an Australian Post Graduate Award and the Yulgilbar Foundation, research support was from the National Health and Medical Research Council of Australia (1098710) and the Australian Research Council (DP180101910).
  This assay was derived and modified from methods originally published in the Journal of cell Biology, Condon et al, 2018.

Competing interests

The authors declare no competing interests.

References

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简介

[ 摘要 ] 细胞表面突起包括富含F-肌动蛋白的波浪状皱纹,这些皱纹是在刺激和细胞迁移过程中短暂竖立的。巨噬细胞是先天性免疫细胞,其组成性地起伏且更具戏剧性 在病原体激活的细胞中。背褶及其产生的巨胞体是环境采样,病原体检测和免疫信号传导的关键部位。波纹的定量评估对于评估巨噬细胞中的病原体反应以及分析其他细胞类型中的生长因子反应非常重要,但是缺乏自动化和定量方法,并且依赖于人工和定性评估。在这里,我们提出了一个自动ImageJ宏,用于量化3D显微镜图像中背侧细胞表面的突起。本文介绍的测定方法适用于高通量筛选应用,可通过测量褶皱的面积和强度并在易于阅读的组合电子表格中提供归一化的值来检测药物,病原体或生长因子引起的细胞褶皱变化。
[背景 ] 细胞在迁移过程中以及对生长因子或其他介质的反应中,会在其表面形成皱纹或直立的膜状膜。作为先天免疫细胞,巨噬细胞在组成上以及在受到生长因子,病原体或其他激活刺激的刺激下都起皱纹。规则可以采取不同的形式。贴壁细胞上表面的背纹通常是直立的。富含F-肌动蛋白的面纱可能是圆形的(因此,其别名为“圆形背褶”)通常会短暂上升,然后塌陷回表面。褶边常形成macropinosomes 如描述在康登等人。,2018 。Macropinosomes 内在皱褶膜,同时摄取流体和可溶的或小的颗粒货物(克尔和蒂斯代尔,2009 ; Commisso 等人。,2013 )。背纹或环状背纹在巨噬细胞,上皮细胞和成纤维细胞中已有较深的描述,它们已被识别为免疫细胞激活的位点(Luo 等,2014; Wall 等,2017),生长因子信号传导(丁二酮)。等人,2004)和活化的受体聚类以及内在化在癌细胞信号传导中起关键作用(Buccione 等人,2004; Orth和McNiven,2006)。许多病原体,包括在病原体进入部位的肠炎沙门氏菌鼠伤寒沙门氏菌,也会引起背褶皱(Ly and Casanova,2007)。

在不同细胞类型上观察到的大的圆形背侧皱纹取决于F-肌动蛋白的瞬时产生,F-肌动蛋白聚合抑制剂如细胞松弛素D消除了它们的形成(Hedberg 等,1993 ;Buccione 等。,2004年)。由于背侧褶皱高度富含F-肌动蛋白,并且比其他富F-actin的投射物(如丝状伪足)在背侧表面上的面积大得多,因此F-actin的荧光标记在背侧褶皱中相对富集。许多研究利用背褶的存在作为细胞反应的定性读出(Gurung 等,2003;Gandhi 等,2004 ;Singla 等,2019)。量化波纹或F-肌动蛋白细胞表面突起的能力,将为从刺激测定到高通量筛选的广泛生物学研究提供有用的分析。通过对全细胞,整个视野的F-肌动蛋白进行测量(Schlam 等人,2016)或通过执行手动选择和感兴趣的用户交互区域(Venter 等人,2015),可以对图像中的波纹进行评估。

这里,我们已经开发了用于背皱褶定量的健壮和自动化测定,需要用于小区选择或两者BA的分割没有用户输入SAL和背区,由核的中点定义。还对Sum-slices图像进行褶皱测量,这意味着要考虑深度信息(即,较高的褶皱会导致强度更高的计数)。该测定法可对多个图像进行批处理,以分析大量细胞,并提供列表结果,以便使用第三方软件轻松进行统计分析。这种强大的自动化分析方法非常适合筛选细胞群,以检测由细胞外刺激(例如病原体,生长因子,激素或细胞因子和/或药理抑制剂,天然产物或营养条件的大规模刺激)引起的背侧波纹的变化。

关键字:Ruffling, 图像分析, F-肌动蛋白, 细胞表面, ImageJ Macro, 自动化, 高通量, 显微镜

材料和试剂


 


用于细胞传代的聚苯乙烯培养皿(Thermo Fisher Scientific,目录号:LBSPD1002X,或等效的)
24孔组织培养板(Thermo Fisher Scientific,目录号:142475,或等效产品)
12毫米1号圆形盖玻片(Thermo Fisher Scientific,目录号:MENCSC121GP或同等产品)
显微镜载玻片26 x 76毫米(VWR,目录号:VWRI631-1558或等效产品)
RAW264.7巨噬细胞样细胞系(ATCC,目录号:TIB071)
透明指甲油(Ultra3,目录号:14111130或同等产品)
RPMI 1640介质(澳大利亚龙萨,目录号:BE12-702F)
胎牛血清(Thermo Fisher Scientific,目录号:16000044)
L-谷氨酰胺100 x (Thermo Fisher Scientific,目录号:25030-081)
4%多聚甲醛(ProSciTech ,目录号:EMS15735-100或同等产品)
海卫一X-100(Sigma - Aldrich,目录号:X100-500ML)
磷酸盐缓冲盐水(PBS)(Thermo Fisher Scientific,目录号:10010023或同等产品)
的AlexaFluor 488鬼笔环肽(Invitrogen,目录号:A12379,见Ñ OTES)
DAPI核染色(Roche公司,目录号:10236276001,或等同物,见Ñ OTES)
ProLong Diamond防褪色试剂(Thermo Fisher Scientific,目录号:P10144,或等效产品)
Poly-D-赖氨酸(Thermo Fisher Scientific,目录号:A3890401或同等产品)
细胞培养基(见食谱)
 


一般


细胞传代耗材
15 ml锥形管
移液器吸头(10、50、100、1,000 微升)
金威普
镜头湿巾
封口膜
纸巾
浸油
卫生署2 Ø
70%乙醇(EtOH)
 


设备


 


自动化血清移液器(Thermo Fisher Scientific,S1移液器填充器,目录号:14-387-163或同等产品)
移液管(P2,P20,P100,P1000)(John Moris ,Gilson#115209或同等学历)
精密镊子(Dumont,样式7,货号:T07-912,或等效产品)
温度和CO 2 控制的培养箱(VWR,目录号:390-2012,或等效产品)
组织培养反相显微镜
血细胞计数器
遮光染色室
注意:甲Ñ铝箔涂布的浅腔室是足够的。


蔡司AxioImager 立式荧光光谱仪显微镜(请参阅“注释”或等效说明)
 


软件


 


Zeiss Zen Blue 2.3(德国卡尔蔡司,或同等的采集软件)
FIJI图像分析程序(Schindelin 等,2012)
Ruffle量化宏(https://github.com/NickCondon/RuffleQuantification)
Microsoft Excel(或等效版本)
GraphPad Prism 8(或等效版本)
 


程序


 


细胞培养
创建您的细胞系选择的悬浮液。
使用血细胞计数器测定细胞浓度,并在组织培养基中稀释至0.1 x 10 6 细胞/ ml 的浓度(配方1)。
将一张盖玻片放入24孔组织板的每个孔中。
加入500μl稀释的细胞悬浮液(0.5×10 5 Ç 厄尔)至每个孔中。
注:d esired汇合第二天= 60%。


用镊子将盖玻片压入井底,以清除盖玻片下方形成的任何气泡。
实验前,让细胞粘附并散布过夜(至少16小时)。
注意:对于更长的治疗或瞬时转染,可能需要降低初始细胞接种密度(固定日的60%融合度是理想的)。


根据需要进行刺激/药理抑制实验。
注意:我们通常使用10-100 ng / μl的脂多糖30分钟以诱导皱纹形成。


吸出培养基并用冰冷的PBS洗涤。
用4%多聚甲醛固定细胞至少30分钟。
用室温PBS三次洗涤细胞。
 


染色盖玻片
将湿纸巾放入染色室的底部。
将胶片放在湿纸巾上。
从24孔板上取下盖玻片,将细胞侧朝上放在封口膜上。
在每个盖玻片上加一滴PBS。
注意:C 滑倒处应始终保持“湿”状态,以确保细胞完整性并防止脱水。


通过在每个盖玻片上滴入0.1 %Triton X-100 滴不超过5分钟来透化细胞。
抽吸用PBS清洗盖玻片3次。
通过将储备溶液分别以1:500和1:1,000稀释到冰冷的PBS中来制备AlexaFluor 488-鬼笔环肽和DAPI(2 mg / ml)标记混合物。
将50μl的标签混合液滴在每个盖玻片附近的石蜡膜上。
从盖玻片上吸出PBS,并将盖玻片倒入标记混合物的液滴上(细胞朝下)。
在避光条件下,于室温(RT,25°C)下孵育至少30分钟。
倒盖玻片并用PBS洗涤3次。
用dH 2 O 清洗盖玻片一次以防止PBS晶体形成。
将一小滴安装介质(我们使用ProLong Diamond)放在显微镜载玻片上。             
注意:为获得最佳结果,在使用之前,应先用70%的乙醇擦拭显微镜载玻片,以除去灰尘和绒毛。


用镊子从旁膜提起盖玻片,轻轻地将盖玻片的边缘靠在干净的Kimwipe纸巾上,除去多余的水。
将盖玻片槽朝下放置在滴落的安装介质上。
轻轻将镊子按在盖玻片的顶部,并吸出多余的安装介质。
用透明的指甲油将盖玻片密封到显微镜载玻片上。
 


影像与数据分析
使用奈奎斯特采样与能够串行光学切片的荧光显微镜,捕获3D Z-堆栈即,Apotome,共焦或宽场系统(图小号1A- 1 C)以捕获细胞和褶边的完整的3维图像。
针对每种情况获取多个视野(每个处理至少收集100个细胞)。
注意:此处的图像是使用Zeiss Axioscan 荧光立式显微镜,使用40 x 1.3 NA Plan Neofluar 物镜和Axiocam 506 CCD相机捕获的。样品用HXP 120W 白光源照明,并用Zeiss DAPI 49 / FITC 44滤光片过滤。Z-叠层物在0.2μm的间隔捕获超出两个可见的顶部和细胞的底部用5 apotome相移用于光学断层识别细胞核和褶边背侧上的中点表面(图小号1D - 1 F) 。


注意:核中点偏移确定。该脚本以其原始形式通过找到横截面积最大的切片来确定原子核的中点(图1G)。在巨噬细胞中,这适当地允许对背褶的选择和分段,这些背褶仅存在于该中点之上,但是对于替代用例或不同的细胞系,可能需要使用不同的选择截止点。图1H显示了最初未通过使用核中点选择外围褶皱的情况,而是通过应用偏移(几个切片)来选择了其他外围褶皱(图1H插图,灰色箭头)。原子核中点偏移值可提供更大的灵活性,并扩大了脚本用于进一步分析应用程序的用途。


                                                                                   


D:\ Reformatting \ 2019-12-16 \ 1902779--1259 Nicholas Condon 689792 \ Figs jpg \ Figure1.jpg


图1. Ruffle量化宏的处理步骤。使用40 x 1.3 NA物镜在此处捕获的宏需要3维2通道图像。图像AC表示Z投影(仅用于图形显示目的)示例图像。A.鬼笔环肽染色的RAW 264.7巨噬细胞(通道1)。B.用DAPI标记的核(通道2)。C.合并两个通道的图像,实心箭头表示重新切割的位置。DF。C的残影(箭头),显示3D图像的X - Z轴(沿面板C中的箭头垂直切开)。D.虚线表示由宏EF选择的原子核的中点。富含F-肌动蛋白的褶皱,带有合并的DAPI,由白色箭头(F)突出显示。比例尺= 20μm。G. 来自原子核的三个不同Z平面的示例横截面图像(X - Y轴)显示了测量面积的差异,最大横截面积被确定为原子核“中点”。H.单元格的剩余部分示例(XZ轴)显示一条实线,确定的核中点在其中,以及如果使用了偏移线(例如,-3偏移),小空心箭头(灰色)突出显示如果使用偏移量将选择的其他褶皱,较大的黑色箭头显示由现有中点切片选择的褶皱,切片之间的差异为偏移量。


 


使用FIJI,安装宏文件:“ Ruffle Analysis Macro”,然后选择“运行”。
注意:要将宏加载到FIJI中,请打开。ijm 文件,或从Github 网站复制文本(请参见上面的软件部分),然后将其粘贴到新的脚本窗口(文件>新建>脚本)中。若要继续使用,请使用文件Ruffle_Analysis_Macro_。ijm 可以复制到FIJI应用程序的“ scripts”文件夹中,下次打开FIJI时,它将出现在插件菜单下。


阅读“确认”屏幕以获取测定说明和说明(图2A)。
导航到要处理的文件的目录位置(图2B)。
在对话框中确认正在使用的文件扩展名(例如,“。czi ”),并根据需要输入Nuclei中点偏移量,然后选择“确定”(图2 C)。
注意:该脚本将在整个目录中运行,仅打开包含所选文件扩展名的图像。


脚本完成后,将出现一个对话框。


使用批处理完成对话框确认宏是否成功运行(图2D)。
导航到输出目录位置(在所选工作目录位置内),并确认阈值选择正在使用输出阈值图像准确选择您感兴趣的区域。
注意:由于宏的简化性质,使用新的或修改的参数重新运行宏所需的时间最少,因此鼓励检查输出数据并在必要时重新运行。


 


D:\ Reformatting \ 2019-12-16 \ 1902779--1259 Nicholas Condon 689792 \ Figs jpg \ Figure2.jpg


图2 。用户交互宏窗口。运行“ Ruffle量化宏”期间需要用户输入的对话框。A.确认和初始屏幕描述宏,创建者的联系方式,输入要求和输出文件。B.与操作系统有关的文件导航器窗口,用于选择包含用于处理和分析的图像的目录。C.文件扩展名过滤器对话框窗口允许用户输入文件扩展名和中点偏移值。D.宏完成窗口通知用户宏的成功完成。


 


Ruffle量化宏的关键步骤概述
使用Otsu算法提取核图像(通道2)并进行阈值处理。通过逐步测量每个切片内的核面积来确定中点。存储具有最大核面积的切片。核数也被计数并存储以供输出(图3A)。
注意:此处使用Otsu阈值化方法来检测原子核,Otsu方法可找到前景和背景之间的类间方差的最大点,适用于预期强度的双峰分布。


F-肌动蛋白(鬼笔环肽)信道(信道1)从图像堆栈中取出成两个单独的图像堆栈,(1)细胞的下半部分取(切片1到细胞核中点切片( 偏移);图3B) (2)取得顶部的褶皱区域(中点切片切片(最偏距 偏移);图3E)。
单元格面积是通过将图像切片(从切片0到确定的核中点切片)进行最大的Z投影确定的(±偏移)和使用Triangle算法进行阈值处理(以选择总的细胞外围)。并测量其输出(图3C)。然后使用距离图来缩小阈值的平坦底部图像,以分离关闭和/或触摸的单元格。然后,使用IJ_IsoData 阈值算法对距离图进行阈值处理,并估计“对象”(单元)的数量并存储以进行输出(图3D )。
注意:此处使用“三角形”算法检测单元格,该算法最适合查找可能仅略高于背景的对象(例如,单元格的最边缘),因此可用于创建准确的选择来测量单元格面积。对于单元格计数,距离映射的阈值单元格会受到Moments IJ_IsoData 阈值的影响,因为它选择了高值最大值(例如,一个或多个非常中心的单元),因此只要精确的单元格数计数就不会受到触摸单元格的影响。


通过对Z投影顶部F-肌动蛋白通道(中点± 偏移至切片总数/顶部的总和)并测量所得的32位图像的强度(乘以检测到的面积)来求和。然后使用矩算法对平坦的顶部/褶皱图像进行阈值化,并测量阈值褶皱的面积(图3F),并用于确定感兴趣区域,以直接测量褶皱强度的总和。
注意:此处使用矩算法对褶皱进行阈值处理,该算法按一系列平均步骤工作,并选择背景以上但强度不同的区域。这有助于从32位总投影图像中准确选择所有褶皱,而不仅仅是最亮的对象。


计算执行normalisations 皱褶区域/小区区域,所述褶边/单元区域的强度。这些值针对检测到的核数进一步标准化。
宏中关键点的图像被保存到输出文件夹(核检测,单元格区域(底部)和褶皱区域(顶部),并且褶皱选择的合并的(绿色/红色)RGB图像覆盖在单元格区域图像上)用于验证的目的(图小号3G- 3 ħ )。
将所有输出值写入日志窗口(图3I ),并将输出.csv文件写入名为Analysis_Results _ < date&time > 的子目录中(图3J)。
来自分析的量化数据存储为.csv文件,其比率和归一化至已计算的细胞数(图3K)。(下面将详细解释值和计算)。
 


 


D:\ Reformatting \ 2019-12-16 \ 1902779--1259 Nicholas Condon 689792 \ Figs jpg \ Figure3.jpg


图3 。褶皱量化宏的处理步骤。一。宏计数的阈值核掩码。公元前。扁平的F-肌动蛋白碱基(底部)和用于测量细胞面积的相应阈值。d 。阈值距离图显示单个细胞分割,用于计数细胞数/视野。EF 。Z投影顶表面(褶皱)的总和,包括用于测量褶皱面积的阈值图像。G. 示例输出图像显示合并的底部(红色)和顶部/皱褶(绿色)。^ h 。示例输出图像显示了合并的阈值底部I (红色)和顶部/褶皱(绿色)I 。“日志”窗口的屏幕快照,其中报告状态,宏运行日期,文件名和结果。J.结果目录的屏幕快照,其中包含输出图像,日志文件和Results.csv文件。K.csv输出结果文件的屏幕快照,其中以下是对源信息的解释,请参见上图(如果适用)。比例尺= 20μm。


 


概述数据处理步骤
输出d ATA (.csv文件)从宏使用开的Microsoft Excel(或等同物)。
原子核中点不正确的数据(例如,切片33/33)表明阈值错误,并且将这些样本的检测排除在分析之外,这可以从相应的阈值输出图像中进一步确认。
将数据复制并分类到相关的组中(基于图像视场和/或处理条件)到GraphPad Prism 8中以进行统计分析。
取决于数据集。统计分析可能包括T检验,单向或双向方差分析。使用内置的绘图工具比较数据。
 


宏的输出和结果
以下详细说明了在输出.csv文件中生成的值的来源:


A列(图像ID)=此数据行的源图像文件名


B列(单元格的估计数)=在图3D中检测到的对象数


C列(核数)=图3A中检测到的物体数


D列(中间切片编号)=原子核中点的图像切片编号


E列(切片总数)=源Z堆栈中的切片数


F列(总细胞面积)=根据图3C 计算得出的面积


G列(总强度)=图3E中的总强度


H列(顶部皱纹)=从图3F 计算得出的面积


第I列(比率顶部:底部)=图3F中的比率面积/图3C中的面积


J列(比率总和强度:像元面积)=比率总和强度图3E /面积图3C


K列(归一化比率)= I列的结果(图3F / 3C )除以C列(图3A 的原子核数)


L列(归一化总和)= J列(图3E / 3C )的结果除以C列(图3A 的原子核数)


 


笔记


 


对于这些实验的蔡司AxioScan 使用显微镜用Apotome 2.以生成荧光图像,但是任何荧光显微镜的光学切片的能力(É 。克,共焦)可以被取代。重要的是,图像必须是3维的,并具有要成像的整个单元格的范围(从底部到最顶部的皱纹)。
在这里,F-肌动蛋白用Alexafluor 488-鬼笔环肽标记,但是任何颜色的鬼笔环肽都可以替代。此外,此宏可与遗传编码的F-肌动蛋白标签(例如GFP-LifeAct)一起使用。核用DAPI染色,但是作为脚本的一部分,其他核/ DNA标记也可能适用于确定核的中点。
对于这些实验,使用了RAW264.7巨噬细胞,但是该方法也已在原代骨髓来源的巨噬细胞(BMM),MB231癌细胞和BV2小胶质细胞上进行了测试。因此,“褶皱定量宏”非常适合用于多种不同的细胞类型,从而可以修改特定的细胞培养技术。
 


菜谱


 


细胞培养基
RPMI 1640培养基


10%Feotal 牛血清


1%聚-D-赖氨酸


 


致谢


 


显微镜在IMB显微镜处进行,澳大利亚癌症研究基金会(ACRF)在IMB处资助了癌症生物学成像设施。NDC获得了澳大利亚研究生奖和Yulgilbar基金会的支持,研究支持来自澳大利亚国家卫生与医学研究委员会(1098710)和澳大利亚研究委员会(DP180101910)


  该测定法是从最初发表于《细胞生物学杂志》(Conden)等人,2018年的方法衍生和修饰的。


 


利益争夺


 


作者宣称没有利益冲突。


 


参考文献


 


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引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Condon, N. D., Stow, J. L. and Wall, A. A. (2020). Automated Analysis of Cell Surface Ruffling: Ruffle Quantification Macro. Bio-protocol 10(2): e3494. DOI: 10.21769/BioProtoc.3494.
  2. Condon, N. D., Heddleston, J. M., Chew, T. L., Luo, L., McPherson, P. S., Ioannou, M. S., Hodgson, L., Stow, J. L. and Wall, A. A. (2018). Macropinosome formation by tent pole ruffling in macrophages. J Cell Biol 217(11): 3873. 
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Annoymous :)
-
Hello! Do all prokaryotes have cell wall?
Is it combination of glycophosphatidylinositol and oligosaccharide is a glycolipid?
Is it lipid-anchored protein is same as glycoprotein?
So, a combination of a glycophosphatidylinositol, oligosaccharide and a protein is a lipid-anchored protein or glycoprotein/glycolipid?
Thanks for advanced!
2020/5/11 1:53:53 回复