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Jun 2021

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Image-based Quantification of Macropinocytosis Using Dextran Uptake into Cultured Cells
基于图像定量使用葡聚糖摄取到培养细胞中的巨胞饮   

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Abstract

Macropinocytosis is an evolutionarily conserved process, which is characterized by the formation of membrane ruffles and the uptake of extracellular fluid. We recently demonstrated a role for CYFIP-related Rac1 Interactor (CYRI) proteins in macropinocytosis. High-molecular weight dextran (70kDa or higher) has generally been used as a marker for macropinocytosis because it is too large to fit in smaller endocytic vesicles, such as those of clathrin or caveolin-mediated endocytosis. Through the use of an image-based dextran uptake assay, we showed that cells lacking CYRI proteins internalise less dextran compared to their wild-type counterparts. Here, we will describe a step-by-step experimentation procedure to detect internalised dextran in cultured cells, and an image pipeline to analyse the acquired images, using the open-access software ImageJ/Fiji. This protocol is detailed yet simple and easily adaptable to different treatment conditions, and the analysis can also be automated for improved processing speed.

Keywords: Macropinocytosis (巨胞饮), Macropinosomes (大胞质体), Dextran (葡聚糖), CYRI-A (CYRI-A), Fam49A (Fam49A), Cell migration (细胞迁移), Actin (肌动蛋白)

Background

Cell migration is one of the many fundamental processes that occur during normal development and physiology (Theveneau and Mayor, 2013; Krause and Gautreau, 2014). Central to this process is the main plasma membrane branched actin generating module, comprising of four main components: the small GTPase protein Rac1 (Machacek et al., 2009), the Scar/WAVE complex (Davidson and Insall, 2011; Krause and Gautreau, 2014), the Arp2/3 complex (Goley and Welch, 2006), and actin. When cells receive a stimulus, such as a growth factor or chemo-attractant, these molecules and complexes work together to nucleate the branched actin network and push the plasma membrane forward, promoting cell migration. Interestingly, macropinocytosis (Swanson and Watts, 1995; Bloomfield and Kay, 2016), an evolutionarily conserved endocytic process, shares the same molecular machinery as cell migration. However, instead of pushing in the plane of the front-rear axis of the cell, local actin polymerisation pushes plasma membrane sheets forward or upward, to form cup-like structures that resolve into macropinocytic vesicles taken into the cell. Cells use macropinocytosis not only to uptake nutrients but also to traffic and organize different membrane receptors, such as integrins, to modulate their adhesion and invasion in cancer (Le et al., 2021).


In our recent paper published in the Journal of Cell Biology (Le et al., 2021), we utilised an image-based internalisation assay, to show that cells lacking CYFIP-related Rac1 Interactor (CYRI) proteins displayed a reduced macropinocytic uptake of dextran 70 kDa.


In this Bio-Protocol article, we describe a step-by-step protocol to detect and quantify the amount of dextran uptake in cultured adherent cells. The wet lab procedure is inspired by Commisso (Commisso et al., 2014), with modifications to simplify the method. We tried different types of dextran, including dextran tetramethylrhodamine (TMR) (Invitrogen, #D1818), dextran Texas Red (Invitrogen, #D1830), and dextran Fluorescein (Invitrogen, #D1822). We found only the last type resulted in clean and analysable data without the need for excessive washing, while both the dextran TMR and dextran Texas Red resulted in a high background with visible clumps of protein, even after multiple washes. We also omit the overnight starving steps, as we found that they were not essential and made no difference to the outcome, using our conditions and cells. This significantly reduced the length of time required for the assay. Since the method described here has been used quite commonly and successfully in the literature before, we focus more on simplifying the experimentation steps and improving the automation capability in the quantification step. We provide the full macro script that can be directly copied and pasted into your Macro window in ImageJ/Fiji (Schindelin et al., 2012). We also provide comments to explain in simple terms what each command in the script does, which could be very useful for those who have no background in coding, or who have just started their journey in programming. We believe this is something that is still missing in the current literature, where there are a lot of resources for image analysis, but many are not necessarily accessible or presented in understandable terms for everyone, particularly beginners. The image analysis pipeline presented here is also suitable for analysing any other intracellular signal, including but not limited to integrin internalisation (Le et al., 2021), transferrin, and other endocytic processes.

Materials and Reagents

  1. Cell culture

    1. Aluminium foil

    2. Parafilm

    3. Paper towel or absorbent tissue

    4. pH test strips, pH-Fix 0–14 PT, fixed indicator (Macherey-Nagel, catalog number: 92111) (optional)

    5. 12-well culture plate (Falcon, catalog number: 353043)

    6. 15-cm tissue culture dish with grid (Fisher Scientific, FalconTM 353025, catalog number: 10314601)

    7. 15-mL conical tubes (Fisher Scientific, FalconTM 352196, catalog number: 11507411)

    8. 19-mm glass coverslips (VWR, catalog number: 631-0156)

    9. COS-7 cells (ATCC, catalog number: CRL-1651)

    10. DMEM (Gibco, catalog number: 21969-035), store at 4°C

    11. L-Glutamine (Gibco, catalog number: 25030-032), store at 4°C

    12. 2.5% Trypsin, no phenol red (Gibco, catalog number: 15090046), store at 4°C

    13. Penicillin-Streptomycin (LifeTechnologies, catalog number: 15140122), store at 4°C

    14. Fetal Bovine Serum (FBS) (Gibco, catalog number: 10270-106), store at 4°C

    15. PE buffer (see Recipes, store at room temperature)


  2. Chemicals

    1. Fibronectin, Bovine Plasma (Sigma-Aldrich, catalog number: F1141), store at 4°C

    2. 16% paraformaldehyde (Electron Microscopy Sciences, catalog number: 15710), store at room temperature

    3. ProLong Diamond antifade mounting medium (Invitrogen, catalog number: P36961), store at -20°C

    4. Hoechst 33342 (Thermo Scientific, catalog number: 62249), store at 4°C

    5. Dextran, Fluorescein, 70,000 MW, Anionic, Lysine Fixable (Invitrogen, catalog number: D1822), store at 4°C, avoid direct light exposure (see Procedure A)

    6. 70% Nitric acid (Sigma-Aldrich, catalog number: 225711), store at room temperature

    7. Ethanol ≥99.8%, (absolute alcohol, without additive) (Sigma-Aldrich, catalog number: 51976), store at room temperature

    8. EDTA (Fisher Scientific, catalog number: 10289410)

    9. Distilled water

    10. PBS buffer tablets (Fisher Scientific, catalog number: 10209252), store at room temperature

    11. PBS buffer (see Recipes)

    12. PE buffer (see Recipes)

    13. Growing DMEM medium (see Recipes)

    14. Serum-free DMEM medium (see Recipes)

    15. 4% PFA solution (see Recipes)

Equipment

  1. Small metal tweezers

  2. A homemade incubating chamber

  3. Haemocytometer or cell counter

Software

  1. Zeiss LSM 710 confocal microscope system (or any other common confocal system if available)

  2. ImageJ or Fiji v2.3.0/1.53m

  3. GraphPad Prism 7

Procedure

The following procedure is based on the experimental conditions that we used to dissect the role of CYRI proteins. A typical experiment should contain at least two incubation timepoints for dextran: 15 and 30 min. Each time point should be done in a separate 12-well plate. Each plate should at least contain one coverslip for scramble control, and one coverslip for each CYRI siRNA-treated sample (minimum of two independent siRNA). You can include other conditions and controls, such as EIPA (a Na+/H+ inhibitor) (Koivusalo et al., 2010), or LY294002 (a PI3-K inhibitor), depending on your experimental setup. The following protocol is described for one coverslip as an example, but this should be scaled up appropriately, depending on your experimental conditions. Any step that deals with cell seeding or matrix coating has to be done inside a biological tissue culture hood. Construction of the incubation chamber and performing the dextran uptake assay can be done outside of the tissue culture hood.


  1. Making an incubating chamber (Figure 1A)

    This step can be done outside of a biological tissue culture hood.

    1. Use a 15-cm tissue culture dish and cover the outer surface of both the plate and the lid with aluminium foil.

    2. Cut a 9 cm × 9 cm piece of parafilm, and place it in the bottom of the dish.

    3. Disinfect the plate with 70% ethanol.



      Figure 1. Making a homemade incubating chamber.

      A. A homemade incubating chamber made out of a 15-cm tissue culture plate covered in aluminium foil. The dotted red line indicates the parafilm placed inside the dish. B. Wet tissue is placed around the edge of the incubating chamber before closing the lid to prevent dehydration of the coated coverslips.


      !NOTE: Construction of the chamber can be done outside of the tissue culture hood. But be sure to spray and wipe the chamber with 70% ethanol, to disinfect it before using it inside a tissue culture hood.


  2. Preparing acid-treated glass coverslips

    This step must be performed in a chemical hood.

    1. Place 19-mm glass coverslips in a 400-mL glass beaker.

    2. Carefully pour 70% nitric acid into the beaker, until all coverslips are submerged.

    3. Very gently swirl the beaker, to allow the acid to make contact with all of the coverslips.

    4. Leave the coverslips in acid for 30 min.

    5. Safely decant the acid back into another bottle (the acid can be reused), and wash the coverslips multiple times with distilled water (50–100 mL each time) for approximately 15 min.

    6. (Optional) Test the pH of the solution, covering the coverslips with a pH strip until it reaches approximately pH 7.0.

    7. Pour the water from the beaker away, and replace it with 100 mL of 70–100% ethanol.

    8. Use parafilm to cover the mouth of the beaker, to slow down evaporation. Store the beaker away from direct sunlight. The coverslips can be safely stored at room temperature, for as long as the ethanol is still present, but note that the ethanol will evaporate over time.

      !NOTE: It is important to wash away all the acid before storing the coverslips in ethanol, as concentrated nitric acid can react with ethanol to form toxic nitric dioxide (NO2) gas.

      !NOTE: If nitric acid is not available, the coverslips can be prepared by standard autoclaving.


  3. Coating coverslips with fibronectin

    This step must be performed inside a biological tissue culture hood.

    1. Disinfect the incubating chamber and tweezers by spraying with 70% ethanol and letting them dry inside a tissue culture hood.

    2. Dilute fibronectin in PBS buffer to a final concentration of 10 μg/mL.

    3. For each coverslip, pipette 40 μL of the fibronectin-PBS solution on top of the parafilm previously placed inside the incubating chamber. The liquid should form a droplet on the parafilm, due to the hydrophobic effect.

    4. Use tweezers to pick up an acid-treated coverslip, quickly wash off the ethanol by dunking it in PBS, and gently place the coverslip on top of the fibronectin droplet in the incubating chamber. Do this for as many coverslips as you require for your experiment.

    5. To prevent dehydration, dampen some tissues with water, place them around the inner edge of the incubating chamber, and make sure they do not touch the coverslips (Figure 1B).

    6. Close the lid, and let the coverslips incubate at room temperature for 1–2 h.

    7. Use tweezers to transfer each coverslip to a well of a 12-well tissue culture plate, and wash them three times with PBS.

    8. Block the coverslips by adding 1 mL of DMEM containing 10% serum to each well. Leave them in the cell incubator (37°C, 5% CO2) until cell seeding.


  4. Seeding cells onto coverslips

    This step must be performed inside a biological tissue culture hood.

    1. From a 10-cm tissue culture plate of COS-7 cells at 80% confluency, aspirate off all of the medium.

    2. Wash the cells with 5 mL of PBS.

    3. Add 300 μL of 0.25% trypsin solution, and incubate in the cell incubator (37°C, 5% CO2) for 5 min.

    4. Add 5 mL of 10%-serum DMEM, to quench the trypsin.

    5. Transfer the cell suspension into a 15-mL Falcon tube, and centrifuge at 500 × g for 5 min.

    6. Aspirate off the medium, and resuspend the cell pellet in 5 mL of 10%-serum DMEM.

    7. Count the cells, either by using a haemocytometer, or an automated cell counter.

    8. Calculate the volume of the cell suspension needed to get 50,000 cells per coverslip, using this equation:

      V is the volume of the cell suspension (μL).

      A is the concentration of cells in the cell suspension (cell/mL).

    9. For each coverslip, make the volume of the 50,000-cell suspension up with the growth medium to 1 mL, and add dropwise to the coverslip.

    10. Incubate the cells at 37°C and 5% CO2 overnight.


  5. Dextran uptake assay

    1. Make up the dextran solution:

      1. Inside a tissue culture hood, resuspend the dextran powder in PBS to the final concentration of 10 mg/mL. Make sure as much dextran is dissolved as possible, by pipetting up and down.

      2. Transfer the content into 1.5-mL Eppendorf tubes, and centrifuge in a tabletop centrifuge at 13,523 × g for 15 min, to get rid of any undissolved dextran.

      3. Without disturbing the pellet, make 50 μL-aliquots of the dextran solution into smaller tubes, and store at -20°C.


    This step can be done outside the tissue culture hood.

    1. On the day of the assay, prepare these before starting the assay:

      1. Cooldown the PBS on ice.

      2. Thaw dextran at room temperature, and make sure it is shielded from any light source.

      3. Warm-up serum-free medium in a 37°C water bath until the experiment.

      4. Prepare an ice tray.

      5. Prepare a 4% paraformaldehyde (PFA) solution in PBS in the chemical fume hood.

      !NOTE: PFA is a COSHH3 chemical and is a known carcinogen, so you must take care when working with it.

    2. Take the two 12-well plates containing your cells that have been seeded on coverslips the day before out of the incubator, and place them on ice.

      !NOTE: Cooling down the cells on ice slows down any endocytic processes and allows cells to be synchronized for when warm dextran solution is added. From our experience, performing this temperature shift gives better internalized-dextran signals.

    3. Aspirate off the medium and wash the cells three times with ice-cold PBS.

    4. Dilute the dextran in a warm serum-free medium, to the final concentration of 0.2 mg/mL.

      !NOTE: The concentration of 0.2 mg/mL was found to be sufficient in our experimental setup. Higher or lower concentrations (0.1–1 mg/mL) can be trialled but, from our experience, 0.2 mg/mL gave the best signal without using too much resources.

    5. Add 1 mL of the dextran solution to each coverslip, and quickly transfer the plates back into the cell incubator for 15 and 30 min, respectively.

    6. After the respective incubation time, wash each plate three times with 1 mL of ice-cold PBS, to stop the endocytosis process and wash away any excess dextran.

    7. In a chemical hood, add 500 μL of 4% PFA to each coverslip to fix the cells at room temperature for 15 min.

    8. Pipette out the PFA and dispose of it appropriately according to your institute regulations. Wash the coverslips three times with PBS.

    9. Dilute Hoechst in PBS to 2 μM, add 50 μL of this solution to each coverslip, and incubate at room temperature in the dark for 30 min.

    10. After 30 min, wash away the excess Hoechst with 1 mL of PBS three times.

    11. Mount the coverslips with 100 μL of mounting medium, and leave them in the dark at room temperature to set overnight.

      !NOTE: The slides can be stored in the dark at 4°C for at least 1 month without much loss of signal. However, we recommend imaging your slides 24 to 36 h after mounting (depending on how fast your mounting media solidifies), to obtain the best results.


  6. Image acquisition

    1. For each coverslip, select ten random fields of view to image.

      !NOTE: To avoid biases and facilitate downstream analyses, the researcher should use the Hoechst channel (305 nm) when selecting the ten random fields of view. Select clusters of cells where the nuclei are evenly distributed all in one focal plane, and avoid places where they are piling on top of each other.

    2. Adjust focus based on the dextran channel (488 nm) and image a single plane with the 40× objective lens of a Zeiss LSM 710 confocal microscope. See Figure 2 for sample images.

      !NOTE: Our samples are flat enough after fixation and mounting, so that a single plane of imaging was sufficient in our case. For thicker samples or imaging at a higher magnification lens, a z-stack with the appropriate interval might be needed. After that, images can be maximally projected into a single image and the same analysis applied.

    3. The Zeiss software automatically exports images under the .czi file format, but .tiff images are also suitable for analysis.



      Figure 2. Example images of the dextran uptake assay of COS-7 cells taken with the Zeiss 710 LSM, 40× lens microscope system, along with an example quantification.

      The control condition is scramble siRNA, and siRNA condition is the combination of both siRNA targeting CYRI-A and CYRI-B. As can be seen, at 15 min of incubation, there is not much difference between the control and the siRNA-treated condition. But after 30 min of incubation, the control cells have taken up significantly more dextran compared to the CYRI-A/CYRI-B double knockdown cells. The quantification graph is adapted from our original publication (Le et al., 2021), with each colour representing data collected from one replication. Error bar = SD. Hoechst = cyan, Dextran = magenta. Scale bars = 20 μm.


  7. Image analysis

    1. Manual analysis (Figure 3)

      1. Open the .czi images (or .tiff) in ImageJ/Fiji.

      2. To separate the dextran and Hoechst channels: Image -> Color -> Split channels.

      3. Convert both channels to 8-bit: Image -> Type -> 8-bit.

      4. Duplicate the dextran channel to analyse the internalised dextran signal in one, and measure the selected area in the other: Image -> Duplicate…

      5. In one of the dextran images, increase the brightness until the outline of the cells becomes visible: Image -> Adjust -> Brightness/Contrast…

      6. Use the Freehand selections tool to outline the selected area for analysis.

      !NOTE: Steps e and f can also be done more easily if cells are also stained with phalloidin (568 nm) to visualise filamentous actin. However, we found this to not be essential in our case.

      !NOTE: Instead of measuring the area covered by cells, we can also count the number of cells in a particular field of view, by counting the number of DAPI-stained nuclei. However, this method should only be used if the cell spread area is relatively unchanged between the different conditions, which is not the case for our CYRI-knockdown cells.

      1. Go to Edit -> Selection -> Make Inverse then press the backspace button to delete the background.

      2. To measure the selected area: Image -> Adjust -> Threshold -> Set to 1, 255. This should highlight the selected area in red.

      3. Analyze -> Analyze Particles -> Set Size: 0–Infinity, Circularity: 0.00–1.00, Show: Outlines, tick Display Results, Summarize and Include holes -> OK.

      4. Transfer the selected region from the first dextran image to the second dextran image by using the combination key: Shift+Command+E on Macbook, then press the backspace button to delete the background.

      !NOTE: For a more general command, you can open the ROI Manager in ImageJ/Fiji: Analyze -> Tools -> ROI Manager… Here you can click Add, to save your selected region from the first dextran image. Then go to the second dextran image, click on the ROI code number of that region in the ROI Manager, the selected region will be transferred. This should work for both Macbook and PC.

      1. To measure the internalised dextran: Image -> Adjust -> AutoThreshold -> MaxEntropy, then analyse particles as described in i.

      2. Copy and paste the result into Excel for further statistical analysis (described in the later section).

      3. Repeat the same procedure for other images.



      Figure 3. Dextran analysis workflow with ImageJ/Fiji.

      The dotted arrow denotes the transferring of the region of interest (magenta outline) from one image to the other by pressing the combination Shift+Command+E.


    2. Semi-automated analysis pipeline using ImageJ/Fiji:

      This section provides the full macro to automate most of the steps described above and can significantly increase the processing speed to at least 100-times faster compared to manual analysis. These macro scripts have been tested, and used successfully for our analysis.

      Before running these macros, make sure to set up your “Analyse Particles” window, as stated in step 1i in the Manual analysis section.


    1. Step 1: Macro for automatically splitting the two channels, adjusting the brightness, and saving the resulting images to a chosen folder.


      Before running the macro, have all original images in a folder of any name. This is where your Source Directory will be.


      Create a second folder of any name outside of the Source Directory. This is where your resulting images will be saved to (Output Directory).


      To open a new Macro window: Plugins -> New -> Macro

      Copy and paste this macro script into a new Macro window.

      !NOTE: When the macro is running, a popup window for each image may appear, depending on the type of image file, click OK to continue.

      An orange colour with “//” in front denotes comments and will not be included in the code when run.

      If you want to know what each of the steps does, simply add the double dashes “//” in front of all of the steps that follow, to exclude them out from the pipeline, or simply copy and paste just one of the command lines into a new Macro window, then click Run.


      //Choose the folder (Source Directory) where your original images are stored.

      selectedFolder = getDirectory("Choose Source Directory ");

      //the getFileList function returns an array of all the file names present in the selectedFolder.

      fileList = getFileList(selectedFolder);

      //the lengthOf function counts the number of files in the fileList array (which, by extension, is in the Source Directory).

      numFiles = lengthOf(fileList);

      //Choose the folder where the exported images are to be saved.

      saveloc = getDirectory("Choose Ouput Directory");

      //This is optional, but you can print out the number of files from the Source Directory folder, as a way of checking if the code is working. A window will pop up, showing the number of files.

      print(numFiles)

      //BatchMode automatically turns off any open image window to avoid cluttering. You can turn this off too.

      setBatchMode(true);

      //This whole block below is an iteration command that will go through each file in the Source Directory, convert the images to 8-bit, adjust their brightness, and save them in your selected folder.

      for (i=0;i<numFiles;i++) {

      file = fileList[i];

      open(selectedFolder + file);

      //Make sure to untick the Split Channels option for multichannel images, in the Bio-Formats Import Option popup window, before the next command. Then, press Enter to continue.

      run("Split Channels");

      selectWindow("C2-" + file);

      run("8-bit");

      run("8-bit");

      rename("1"); //Rename the image to easier select window later on

      run("Duplicate...", " ");

      selectWindow("1-1");

      run("Brightness/Contrast...");

      setMinAndMax(0, 20);

      //Save images with a prefix “Area_” or “Dextran_” appended to the original file name.

      saveAs("Tiff", saveloc + "Area_" + file);

      selectWindow("1");

      saveAs("Tiff", saveloc + "Dextran_" + file);

      run("Close All");

      }

    2. Step 2:

      This is the only manual step.

      Now that all your processed images are saved into your output folder, open the images with “Area_” prefix in their name, repeat steps 1f and 1g in the Manual analysis section, and use step 1j to transfer the selected region of interest from the “Area_” images to the images with “Dextran_” prefix in their name.

      Save the newly processed images into a new folder called “Area” and “Dextran”, respectively.

      If phalloidin staining was included, cells can be automatically segmented and this step can be automated.

      !NOTE: The saving step can be automated too. You can record the steps using a macro recording window: Plugins -> Macro -> Record…


    1. Step 3:

      Macro for measuring the area of the selected region.

      Copy and paste this macro into a new Macro window and click Run.

      When this macro finishes running, a Results file containing the measurements will be saved to the selected Output Directory.


      //Choose the “Area” folder as your Source Directory.

      selectedFolder = getDirectory("Choose Source Directory");


      fileList = getFileList(selectedFolder);


      numFiles = lengthOf(fileList);


      //Choose the same “Area” folder as your Output Directory.

      Saveloc = getDirectory("Choose Output Directory");


      setBatchMode(true);


      //This whole block below is an iteration command that will go through each file in the Source Directory, measure the area of the selected region, and export the results as a csv. file to the “Area” file.

      for (i=0;i<numFiles;i++) {

      file = fileList[i]; //Indexing the file from 0 to i.

      open(selectedFolder + file);

      setAutoThreshold("Default dark");

      run("Threshold...");

      setThreshold(1, 250);

      run("Analyze Particles...", " show=Outlines display summarize");

      selectWindow("Results"); //This selects the Results window

      //This saves the Results file to the folder called "Area"

      saveAs("Results", "/Users/ale/Desktop/Area/Results.csv");

      run("Close All");

      }


    2. Step 4:

      Macro for measuring the internalizedsed dextran.

      Copy and paste this macro into a new Macro window and click Run.

      When this macro finishes running, a Summary file containing the measurements will be saved to the selected Output Directory.


      //Choose the “Dextran” folder as your Source Directory.

      selectedFolder = getDirectory("Choose Source Directory");


      fileList = getFileList(selectedFolder);


      numFiles = lengthOf(fileList);


      //Choose the same “Dextran” folder as your Output Directory.

      saveloc = getDirectory("Choose Output Directory");


      setBatchMode(true);


      //This whole block below is an iteration command that will go through each file in the Source Directory, measure the internalizedsed Dextran signal, and export the Summary file as a csv. file to the Dextran folder.

      for (i=0;i<numFiles;i++) {

      file = fileList[i];

      open(selectedFolder + file);

      //Apply MaxEntropy AutoThreshold to images

      //You can use a different AutoThreshold other than MaxEntropy, depending on your image acquisition.

      run("Auto Threshold", "method=MaxEntropy white");

      run("Threshold...");

      setThreshold(255, 255);

      run("Analyze Particles...", " show=Outlines display summarize");

      run("Close All");

      }

      selectWindow("Summary");

      saveAs("Results", "/Users/ale/Desktop/Dextran/Summary.csv");

      //The last two commands are outside the "for" loop, to allow all the Summary results to be put into the same window and therefore saved all at once.

    Data analysis

    Once you have analysed all of the images and obtained the area of the region of interest, as well as the area of the dextran signal, we can calculate the internalisation index by using the formula:



    Plot the internalisation index using the appropriate software, such as Graphpad Prism. At least three independent biological replicates (separate experiments are repeated at least three times on three separate days) are performed before analysing. You can also include technical replicates for each tested condition within each biological replicate, based on your hypothesis and experimental design.

    If comparing between two groups, inspect if their distribution is normally distributed, and then apply a two-tailed two-sample unpaired t-test.

    The description of our analysis can be found in the Statistical Analysis section, as well as the legend of Figure 5 in our original manuscript (Le et al., 2021).


    For testing out the macro, please visit Anh Hoang Le’s Github page: https://github.com/AnhLe2702/20211019_MacroImageJ_Automatic_split_export_images and download two test image files, labelled scr_30_6.czi and 36_30_6.czi.

    Recipes

    1. PBS buffer

      1 L of PBS contains 10 PBS tablets in 1 L of distilled water.

    2. PE buffer

      5 L of PBS buffer, 1.86 g of EDTA.

    3. Growing DMEM medium

      500 mL of DMEM, 50 mL of serum, 5 mL of L-Glutamine, 5 mL of Penicillin-Streptomycin.

    4. Serum-free DMEM medium

      500 mL of DMEM, 5 mL of L-Glutamine, 5 mL of Penicillin-Streptomycin.

    5. 4% PFA solution

      10 mL of 16% PFA

      30 mL of PBS

    Acknowledgments

    We thank Cancer Research UK for core funding (A17196 and A31287) and funding to L.M. Machesky (A24452).

    The wet lab procedure was inspired by Commisso et al. (2014) and Anh H. Le et al. (2021) .

    Le, A. H., Yelland, T., Paul, N. R., Fort, L., Nikolaou, S., Ismail, S. and Machesky, L. M. (2021). CYRI-A limits invasive migration through macropinosome formation and integrin uptake regulation. J Cell Biol 220(9).

    Competing interests

    The authors declare no competing interests.

    References

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    2. Commisso, C., Flinn, R. J. and Bar-Sagi, D. (2014). Determining the macropinocytic index of cells through a quantitative image-based assay. Nat Protoc 9(1): 182-192.
    3. Davidson, A. J. and Insall, R. H. (2011). Actin-based motility: WAVE regulatory complex structure reopens old SCARs. Curr Biol 21(2): R66-68.
    4. Goley, E. D. and Welch, M. D. (2006). The ARP2/3 complex: an actin nucleator comes of age. Nat Rev Mol Cell Biol 7(10): 713-726.
    5. Koivusalo, M., Welch, C., Hayashi, H., Scott, C. C., Kim, M., Alexander, T., Touret, N., Hahn, K. M. and Grinstein, S. (2010). Amiloride inhibits macropinocytosis by lowering submembranous pH and preventing Rac1 and Cdc42 signaling. J Cell Biol 188(4): 547-563.
    6. Krause, M. and Gautreau, A. (2014). Steering cell migration: lamellipodium dynamics and the regulation of directional persistence. Nat Rev Mol Cell Biol 15(9): 577-590.
    7. Le, A. H., Yelland, T., Paul, N. R., Fort, L., Nikolaou, S., Ismail, S. and Machesky, L. M. (2021). CYRI-A limits invasive migration through macropinosome formation and integrin uptake regulation. J Cell Biol 220(9): e202012114.
    8. Machacek, M., Hodgson, L., Welch, C., Elliott, H., Pertz, O., Nalbant, P., Abell, A., Johnson, G. L., Hahn, K. M. and Danuser, G. (2009). Coordination of Rho GTPase activities during cell protrusion. Nature 461(7260): 99-103.
    9. Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T., Preibisch, S., Rueden, C., Saalfeld, S., Schmid, B., et al., A. (2012). Fiji: an open-source platform for biological-image analysis. Nat Methods 9(7): 676-682.
    10. Swanson, J. A. and Watts, C. (1995). Macropinocytosis. Trends Cell Biol 5(11): 424-428.
    11. Theveneau, E. and Mayor, R. (2013). Collective cell migration of epithelial and mesenchymal cells. Cell Mol Life Sci 70(19): 3481-3492.

简介

摘要:巨胞饮作用是一个进化上保守的过程,其特征在于膜褶皱的形成和细胞外液的吸收。我们最近证明了 CYFIP 相关 Rac1 相互作用 (CYRI) 蛋白在巨胞饮作用中的作用。高分子量葡聚糖(70kDa 或更高)通常被用作巨胞饮作用的标志物,因为它太大而无法适应较小的内吞囊泡,例如网格蛋白或小窝蛋白介导的内吞作用。通过使用基于图像的葡聚糖摄取测定,我们发现与野生型对应物相比,缺乏 CYRI 蛋白的细胞内化的葡聚糖更少。在这里,我们将描述一个分步实验程序,以检测培养细胞中的内化葡聚糖,以及使用开放访问软件 ImageJ/Fiji 分析所获得图像的图像管道。该协议详细而简单,易于适应不同的治疗条件,分析也可以自动化以提高处理速度。


背景

細胞遷移是正常發育和生理過程中發生的許多基本過程之一(Theveneau 和 Mayor,2013;Krause 和 Gautreau,2014) 。該過程的核心是主要的質膜分支肌動蛋白生成模塊,由四個主要成分組成:小 GTPase 蛋白 Rac1 (Machacek等人,2009) 、Scar/WAVE 複合物(Davidson 和 Insall,2011;Krause 和 Gautreau, 2014) 、Arp2/3 複合體(Goley and Welch, 2006)和肌動蛋白。當細胞受到刺激時,例如生長因子或化學引誘劑,這些分子和復合物共同作用,使分支的肌動蛋白網絡成核並推動質膜向前移動,從而促進細胞遷移。有趣的是,巨胞飲作用 (Swanson and Watts, 1995; Bloomfield and Kay, 2016)是一種進化上保守的內吞過程,與細胞遷移具有相同的分子機制。然而,局部肌動蛋白聚合不是推動細胞的前後軸平面,而是向前或向上推動質膜片,形成杯狀結構,分解成進入細胞的大胞飲囊泡。 細胞利用巨胞飲作用不僅可以吸收營養,還可以運輸和組織不同的膜受體,例如整合素,以調節它們在癌症中的粘附和侵襲 (Le等人,2021 年) 。
在我們最近發表在《細胞生物學雜誌》 ( Le et al. , 2021 )上的論文中,我們利用基於圖像的內化分析表明,缺乏 CYFIP 相關 Rac1 相互作用 (CYRI) 蛋白的細胞顯示出減少的葡聚醣巨胞飲攝取70 kDa 。
在這篇生物協議文章中,我們描述了一個分步協議來檢測和量化培養的貼壁細胞中葡聚醣的攝取量。濕實驗室程序的靈感來自Commisso (Commisso等人,2014 年) ,進行了修改以簡化方法。我們嘗試了不同類型的葡聚醣,包括葡聚醣四甲基羅丹明(TMR)(Invitrogen,#D1818)、葡聚醣德克薩斯紅(Invitrogen,#D1830)和葡聚醣熒光素(Invitrogen,#D1822)。我們發現只有最後一種類型可以產生乾淨和可分析的數據,而無需過度洗滌,而葡聚醣 TMR 和葡聚醣德克薩斯紅都產生高背景,即使在多次洗滌後也有可見的蛋白質團塊。我們還省略了隔夜挨餓的步驟,因為我們發現它們不是必需的,對結果沒有影響,使用我們的條件和細胞。這顯著減少了測定所需的時間長度。由於這裡描述的方法在之前的文獻中已經非常普遍和成功地使用,我們更關注簡化實驗步驟和提高量化步驟的自動化能力。我們提供完整的宏腳本,可以直接複製並粘貼到 ImageJ/Fiji 的宏窗口中(Schindelin等人,2012 年) 。我們還提供註釋以簡單地解釋腳本中的每個命令的作用,這對於那些沒有編碼背景或剛剛開始編程之旅的人可能非常有用。我們相信這是當前文獻中仍然缺少的東西,其中有很多用於圖像分析的資源,但許多資源不一定可以訪問或以每個人都可以理解的方式呈現,尤其是初學者。這裡介紹的圖像分析管道也適用於分析任何其他細胞內信號,包括但不限於整合素內化 (Le et al. , 2021) 、轉鐵蛋白和其他內吞過程。

关键字:巨胞饮, 大胞质体, 葡聚糖, CYRI-A, Fam49A, 细胞迁移, 肌动蛋白

材料和試劑
細胞培養
鋁箔_
封口膜
紙巾或吸水紙巾
pH試紙,pH-Fix 0-14 PT,固定指示劑( Macherey -Nagel,目錄號:92111)(可選)
12孔培養板(Falcon,目錄號:353043)
帶網格的15厘米組織培養皿(Fisher Scientific, Falcon TM 353025,目錄號:10314601)
15-mL錐形管(Fisher Scientific, Falcon TM 352196,目錄號:11507411)
19 - mm玻璃蓋玻片( VWR ,目錄號: 631-0156 )
COS-7 電池(ATCC,目錄號:CRL-1651)
DMEM(Gibco,目錄號:21969-035),4°C 儲存
L-谷氨酰胺(Gibco,目錄號:25030-032),儲存於 4°C
2.5% 胰蛋白酶,不含酚紅(Gibco,目錄號:15090046),4°C 儲存
青黴素-鏈黴素( LifeTechnologies ,目錄號:15140122),4°C儲存
胎牛血清(FBS)(Gibco,目錄號:10270-106),4°C儲存
PE 緩衝液(參見配方,室溫儲存)


化學品
纖連蛋白,牛血漿(Sigma-Aldrich,目錄號:F1141),在4°C下儲存
16%多聚甲醛(Electron Microscopy Sciences,目錄號:15710),室溫儲存
ProLong Diamond 抗褪色封固劑(Invitrogen,目錄號:P36961),儲存於-20 °C
Hoechst 33342(Thermo Scientific,目錄號:62249),儲存於 4°C
葡聚醣,熒光素,70,000 MW,陰離子,賴氨酸可固定(Invitrogen,目錄號:D1822),儲存在 4 °C,避免直接光照(參見程序 A)
70%硝酸(Sigma-Aldrich,目錄號:225711),室溫儲存
乙醇≥99.8%,(無添加劑,無添加劑)(Sigma-Aldrich,目錄號:51976),室溫儲存
EDTA(Fisher Scientific,目錄號:10289410)
蒸餾水
PBS緩沖片(Fisher Scientific,目錄號:10209252),室溫儲存
PBS 緩衝液(見配方)
PE 緩衝液(見配方)
生長 DMEM 培養基(見食譜)
無血清 DMEM 培養基(見配方)
4% PFA 溶液(見配方)




設備


小金屬鑷子
自製孵化室
血細胞計數器或細胞計數器




軟件


Zeiss LSM 710 共聚焦顯微鏡系統(或任何其他常見的共聚焦系統,如果有)
ImageJ 或斐濟 v2.3.0/1.53m
GraphPad 棱鏡 7




程序


以下程序基於我們用來剖析 CYRI 蛋白作用的實驗條件。一個典型的實驗應包含至少兩個葡聚醣的孵育時間點:15 分鐘和 30 分鐘。每個時間點應在單獨的 12 孔板中完成。每個板應至少包含一個用於加擾控制的蓋玻片,每個 CYRI siRNA 處理的樣品應包含一個蓋玻片(至少兩個獨立的 siRNA)。您可以包括其他條件和對照,例如 EIPA(一種 Na + /H +抑製劑) (Koivusalo等人,2010 年)或 LY294002(一種 PI3-K 抑製劑),具體取決於您的實驗設置。以下協議以一個蓋玻片為例進行了描述,但應根據您的實驗條件適當放大。任何處理細胞播種或基質塗層的步驟都必須在生物組織培養罩內完成。可以在組織培養罩外進行孵育室的構建和葡聚醣攝取測定。


製作孵化室 (圖1A)


此步驟可以在生物組織培養罩之外完成。
使用 15 厘米的組織培養皿,並用鋁箔覆蓋板和蓋子的外表面。
剪下一塊 9 cm × 9 cm 的封口膜,放在盤子底部。
用 70% 乙醇對盤子進行消毒。


 


圖1 。製作自製的孵化室。 
A. 自製的培養室,由15 厘米的組織培養板覆蓋鋁箔製成。紅色虛線表示放置在盤內的封口膜。 B. 在蓋上蓋子之前,將濕紙巾放在培養室的邊緣周圍,以防止塗層蓋玻片脫水。


 


準備酸處理的玻璃蓋玻片


此步驟必須在化學罩中進行。
將 19 毫米玻璃蓋玻片放入 400 毫升玻璃燒杯中。
小心地將 70% 的硝酸倒入燒杯中,直到所有蓋玻片都被淹沒。
非常輕輕地旋轉燒杯,讓酸與所有蓋玻片接觸。
將蓋玻片放在酸中 30 分鐘。
安全地將酸倒回另一個瓶子(酸可以重複使用),並用蒸餾水(每次 50-100 mL)多次清洗蓋玻片約15 分鐘。
(可選)測試溶液的 pH 值,用 pH 條覆蓋蓋玻片,直到其達到大約 pH 7.0。
將燒杯中的水倒掉,並用 100 mL 的 70–100% 乙醇代替。
用封口膜蓋住燒杯口,以減緩蒸發。將燒杯存放在避免陽光直射的地方。只要乙醇仍然存在,蓋玻片就可以在室溫下安全儲存,但請注意乙醇會隨著時間的推移而蒸發。


 


用纖連蛋白包被蓋玻片


此步驟必須在生物組織培養罩內進行。
通過噴灑 70% 乙醇並讓它們在組織培養罩內乾燥,對孵化室和鑷子進行消毒。
將 PBS 緩衝液中的纖連蛋白稀釋至最終濃度為 10 μg / mL 。
對於每個蓋玻片,將 40 μL的纖連蛋白-PBS 溶液移液到先前放置在孵化室內的封口膜頂部。由於疏水作用,液體應在封口膜上形成液滴。
使用鑷子拿起酸處理的蓋玻片,通過將其浸入 PBS 中快速洗掉乙醇,然後將蓋玻片輕輕放在孵育室中纖連蛋白液滴的頂部。為您的實驗所需的盡可能多的蓋玻片執行此操作。
為了防止脫水,用水潤濕一些組織,將它們放在孵化室的內邊緣,並確保它們不接觸蓋玻片(圖1B )。
蓋上蓋子,讓蓋玻片在室溫下孵育 1-2 小時。
使用鑷子將每個蓋玻片轉移到 12 孔組織培養板的孔中,並用 PBS 洗滌 3 次。
通過在每口井中添加 1 mL 含有 10% 血清的 DMEM 來阻止蓋玻片。將它們留在細胞培養箱中(37 °C,5% CO 2 ) ,直到細胞播種。


將細胞播種到蓋玻片上


此步驟必須在生物組織培養罩內進行。
從 80% 融合的 COS-7 細胞的 10 厘米組織培養板中吸出所有培養基。
用 5 mL 的 PBS 清洗細胞。
加入 300 μL的 0.25% 胰蛋白酶溶液,在細胞培養箱(37 °C,5% CO 2 )中孵育 5 分鐘。
添加 5 mL 的 10% 血清 DMEM,以淬滅胰蛋白酶。
將細胞懸液轉移到 15 mL Falcon 管中,並以 500 × g離心5 分鐘。
吸出培養基,將細胞顆粒重新懸浮在 5 mL 的 10% 血清 DMEM 中。
使用血細胞計數器或自動細胞計數器對細胞進行計數。
使用以下等式計算每個蓋玻片獲得 50,000 個細胞所需的細胞懸浮液體積:


V=  50,000/A×1000 (μL)
V 是細胞懸液的體積( μL )。
A 是細胞懸液中的細胞濃度(細胞/mL)。
對於每個蓋玻片,將 50,000 細胞懸浮液的體積與生長培養基一起增加到 1 mL,然後逐滴添加到蓋玻片中。
37 °C 和 5% CO 2下孵育細胞過夜。


葡聚醣攝取測定


配製葡聚醣溶液:
在組織培養罩內,將 PBS 中的葡聚醣粉末重新懸浮至最終濃度為 10 mg/ mL。通過上下移液確保盡可能多的葡聚醣溶解。
將內容物轉移到 1.5 mL Eppendorf 管中,在台式離心機中以 13,523 × g離心15 分鐘,以除去任何未溶解的葡聚醣。
在不干擾沉澱的情況下,將 50 μL的葡聚醣溶液等分到較小的試管中,並在 -20°C 下儲存。


此步驟可以在組織培養罩外完成。
在化驗當天,在開始化驗前準備好這些:
在冰上冷卻 PBS。
在室溫下解凍葡聚醣,並確保它不受任何光源的影響。
37 °C 水浴中預熱無血清培養基,直到實驗。
準備一個冰盤。
在化學通風櫃中的 PBS 中製備 4% 的多聚甲醛 (PFA) 溶液。


 


將前一天接種在蓋玻片上的兩個 12 孔板從培養箱中取出,放在冰上。


 


吸出培養基並用冰冷的 PBS 洗滌細胞 3 次。
在溫暖的無血清培養基中稀釋葡聚醣,最終濃度為 0.2 毫克/毫升。


 


在每個蓋玻片中加入 1 mL 的葡聚醣溶液,然後將板分別快速轉移回細胞培養箱中 15 分鐘和 30 分鐘。
在各自的孵育時間後,用 1 mL 的冰冷 PBS 清洗每個板 3 次,以停止內吞過程並洗去任何多餘的葡聚醣。
在化學罩中,將 500 μL的 4% PFA 添加到每個蓋玻片中,以在室溫下將細胞固定 15 分鐘。
移出 PFA 並根據您所在機構的規定進行適當處理。用 PBS 清洗蓋玻片 3 次。
PBS 中的 Hoechst 稀釋至 2 μM ,在每個蓋玻片中加入 50 μL該溶液,並在室溫下避光孵育 30 分鐘。
30 分鐘後,用 1 mL 的 PBS 洗去多餘的 Hoechst 三次。
μL的安裝介質安裝蓋玻片,並在室溫下將其置於黑暗中過夜。


 


圖像採集


對於每個蓋玻片,選擇十個隨機視野進行成像。


 


根據葡聚醣通道 (488 nm) 調整焦點,並使用蔡司 LSM 710 共聚焦顯微鏡的 40 倍物鏡對單個平面進行成像。有關示例圖像,請參見圖2 。


 


圖2 。使用 Zeiss 710 LSM、40 ×透鏡顯微鏡系統拍攝的 COS-7 細胞的葡聚醣攝取測定示例圖像,以及示例量化。
對照條件是scramble siRNA,siRNA條件是靶向CYRI-A和CYRI-B的siRNA的組合。可以看出,在孵育 15 分鐘時,對照和 siRNA 處理的條件之間沒有太大差異。但在孵育 30 分鐘後,與 CYRI-A/CYRI-B 雙敲低細胞相比,對照細胞吸收了更多的葡聚醣。量化圖改編自我們的原始出版物 (Le et al ., 2021),每種顏色代表從一次復制中收集的數據。誤差線 = SD。 Hoechst = 青色,葡聚醣 = 洋紅色。比例尺 = 20 μm 。


 


蔡司軟件自動導出 . czi文件格式,但 .tiff 圖像也適合分析。


圖像分析


人工分析(圖 3 )
打開. ImageJ/Fiji 中的czi圖像(或 .tiff)。
要分離 dextran 和 Hoechst 通道:圖像 -> 顏色 -> 拆分通道。
將兩個通道都轉換為 8 位:圖像 -> 類型 -> 8 位。
複製葡聚醣通道以分析一個內化的葡聚醣信號,並測量另一個中的選定區域:圖像 -> 複製...
在其中一張葡聚醣圖像中,增加亮度直到單元格的輪廓變得可見:圖像 -> 調整 -> 亮度/對比度...
使用手繪選擇工具勾勒選定區域以進行分析。


 


轉到編輯 -> 選擇 -> 反轉,然後按退格鍵刪除背景。
要測量選定區域:圖像 -> 調整 -> 閾值 -> 設置為 1, 255。這應該以紅色突出顯示選定區域。
分析 -> 分析粒子 -> 設置大小:0–Infinity,圓度:0.00–1.00,顯示:輪廓,勾選顯示結果,匯總並包含孔 -> 確定。
使用組合鍵將選定區域從第一個葡聚醣圖像轉移到第二個葡聚醣圖像: Macbook上的Shift+Command+E ,然後按退格鍵刪除背景。


 


要測量內化的葡聚醣: Image -> Adjust -> AutoThreshold -> MaxEntropy ,然後如i中所述分析粒子。
將結果復制並粘貼到 Excel 中以進行進一步的統計分析(在後面的部分中描述)。
對其他圖像重複相同的過程。




 


圖3 。使用 ImageJ/Fiji 的葡聚醣分析工作流程。 
虛線箭頭表示通過按下組合Shift+Command+E將感興趣區域(洋紅色輪廓)從一個圖像轉移到另一個圖像。


使用 ImageJ/Fiji 的半自動分析管道:
本節提供完整的宏來自動執行上述大部分步驟,並且與手動分析相比,可以將處理速度顯著提高至少 100 倍。這些宏腳本已經過測試,並成功用於我們的分析。
在運行這些宏之前,請確保設置您的“分析粒子”窗口,如手動分析部分的步驟 1i 中所述。


第 1 步:自動分割兩個通道、調整亮度並將生成的圖像保存到所選文件夾的宏。


在運行宏之前,將所有原始圖像放在任意名稱的文件夾中。這是您的源目錄所在的位置。


在源目錄之外創建第二個任意名稱的文件夾。這是您生成的圖像將保存到(輸出目錄)的位置。


打開一個新的宏窗口:插件 -> 新建 -> 宏
將此宏腳本複制並粘貼到新的宏窗口中。


 


前面帶“//”的橙色表示註釋,運行時不會包含在代碼中。
如果您想知道每個步驟的作用,只需在隨後的所有步驟前添加雙破折號“//”,將它們從管道中排除,或者只需複制並粘貼其中一個命令行進入一個新的宏窗口,然後單擊運行。


//選擇存儲原始圖像的文件夾(源目錄)。
selectedFolder = getDirectory ( "選擇源目錄");


// getFileList函數返回selectedFolder中存在的所有文件名的數組。
fileList = getFileList ( selectedFolder );


// lengthOf函數計算fileList數組中的文件數(通過擴展,它位於源目錄中)。
numFiles = lengthOf ( fileList );


//選擇要保存導出圖像的文件夾。
saveloc = getDirectory ( "選擇輸出目錄");


//這是可選的,但您可以打印出源目錄文件夾中的文件數量,作為檢查代碼是否正常工作的一種方式。將彈出一個窗口,顯示文件的數量。
打印(數字文件)


// BatchMode自動關閉任何打開的圖像窗口以避免混亂。您也可以將其關閉。
設置批處理模式(真);


//下面的整個塊是一個迭代命令,它將遍歷源目錄中的每個文件,將圖像轉換為 8 位,調整它們的亮度,並將它們保存在您選擇的文件夾中。
對於 ( i = 0;i < numFiles;i ++) {
文件=文件列表[我] ;
打開(選定文件夾+文件);
//確保在下一個命令之前在 Bio-Formats Import Option 彈出窗口中取消選中多通道圖像的 Split Channels 選項。然後,按 Enter 繼續。
運行( “拆分頻道”);
選擇窗口( “C2-”+文件);
運行(“8位”);
運行(“8位”);
重命名( “1”); //將圖像重命名為稍後更容易選擇的窗口
運行( “重複...”,“”);
選擇窗口( “1-1”);
run( "亮度/對比度...");
setMinAndMax ( 0, 20);
//保存帶有前綴“Area_”或“Dextran_”的圖像,附加到原始文件名。
saveAs ( “Tiff”, saveloc +“Area_”+文件);
選擇窗口( “1”);
saveAs ( "Tiff", saveloc + "Dextran_" + 文件);
運行( “關閉所有”);
}


第2步:
這是唯一的手動步驟。
現在所有處理過的圖像都保存到輸出文件夾中,打開名稱中帶有“Area_”前綴的圖像,在手動分析部分重複步驟 1f 和 1g,然後使用步驟 1j 從“ Area_”圖像到名稱中帶有“Dextran_”前綴的圖像。
將新處理的圖像分別保存到名為“Area”和“Dextran”的新文件夾中。
如果包括鬼筆環肽染色,可以自動分割細胞,這一步可以自動化。


 


第 3 步:
用於測量所選區域面積的宏。
將此宏複製並粘貼到新的宏窗口中,然後單擊運行。
當這個宏完成運行時,包含測量結果的結果文件將保存到選定的輸出目錄中。


//選擇“區域”文件夾作為您的源目錄。
selectedFolder = getDirectory ( "選擇源目錄");


fileList = getFileList ( selectedFolder );


numFiles = lengthOf ( fileList );


//選擇與輸出目錄相同的“區域”文件夾。
Saveloc = getDirectory ( "選擇輸出目錄");


設置批處理模式(真);


//下面這整個塊是一個迭代命令,它將遍歷源目錄中的每個文件,測量所選區域的面積,並將結果導出為 csv。文件到“區域”文件。
對於 ( i = 0;i < numFiles;i ++) {
文件=文件列表[我] ; //將文件從 0 索引到i 。
打開(選定文件夾+文件);
setAutoThreshold ( "默認暗");
運行( “閾值...”);
設置閾值( 1, 250);
run( "分析粒子...", "show=Outlines 顯示總結");
選擇窗口(“結果”); //這會選擇結果窗口
//這會將結果文件保存到名為“Area”的文件夾中
saveAs ( “結果”,“/Users/ale/Desktop/Area/Results.csv”);
運行( “關閉所有”);
}


第四步:
用於測量內化葡聚醣的宏。
將此宏複製並粘貼到新的宏窗口中,然後單擊運行。
當這個宏完成運行時,包含測量結果的摘要文件將保存到選定的輸出目錄中。


//選擇“Dextran”文件夾作為您的源目錄。
selectedFolder = getDirectory ( "選擇源目錄");


fileList = getFileList ( selectedFolder );


numFiles = lengthOf ( fileList );


//選擇與輸出目錄相同的“Dextran”文件夾。
saveloc = getDirectory ( "選擇輸出目錄");


設置批處理模式(真);


//下面的整個塊是一個迭代命令,它將遍歷源目錄中的每個文件,測量內化的葡聚醣信號,並將摘要文件導出為 csv。文件到葡聚醣文件夾。
對於 ( i = 0;i < numFiles;i ++) {
文件=文件列表[我] ;
打開(選定文件夾+文件);
//應用最大熵 圖像的自動閾值
//您可以使用除 MaxEntropy 之外的其他AutoThreshold ,具體取決於您的圖像採集。
運行( “自動閾值”,“方法=最大熵白色”);
運行( “閾值...”);
設置閾值( 255、255);
run( "分析粒子...", "show=Outlines 顯示總結");
運行( “關閉所有”);
}
選擇窗口(“摘要”);
saveAs ( “結果”,“/Users/ale/Desktop/Dextran/Summary.csv”);
//最後兩個命令在“for”循環之外,以允許將所有摘要結果放入同一個窗口,從而一次保存所有結果。


數據分析


一旦你分析了 所有圖像並獲得感興趣區域的面積,以及葡聚醣信號的面積,我們可以使用公式計算內化指數:


Internalisation index=  (Dextran signal)/(Area of region of interest)


使用適當的軟件(例如Graphpad Prism )繪製內化指數。在分析之前進行至少三個獨立的生物學重複(單獨的實驗在三天內至少重複三次) 。您還可以根據您的假設和實驗設計,在每個生物複製中包含每個測試條件的技術複製。
如果在兩組之間進行比較,檢查它們的分佈是否為正態分佈,然後應用雙尾雙樣本非配對 t 檢驗。
我們的分析描述可以在統計分析部分找到,以及我們原始手稿中圖 5 的圖例( Le et al. , 2021 ) 。


如需測試宏,請訪問 Anh Hoang Le's Github頁面: https ://github.com/AnhLe2702/20211019_MacroImageJ_Automatic_split_export_images並下載兩個測試圖像文件,分別標記為 scr_30_6.czi 和 36_30_6.czi。




食譜


PBS緩衝液
1 L PBS 在 1 L 蒸餾水中含有 10 片 PBS。


PE緩衝液
5 L PBS 緩衝液,1.86 g EDTA。


生長 DMEM 培養基
500 mL DMEM、50 mL血清、5 mL L-谷氨酰胺、5 mL青黴素-鏈黴素。


無血清 DMEM 培養基
500 mL DMEM、5 mL L-谷氨酰胺、5 mL 青黴素-鏈黴素。


4% PFA 溶液
10 毫升 16% PFA
30 毫升 PBS




致謝


我們感謝英國癌症研究中心的核心資金(A17196 和 A31287)以及對 LM Machesky的資金(A24452)。
濕實驗室程序的靈感來自Commisso 等人。 (2014) 和 Anh H. Le等人。 (2021 年)。


Le, AH, Yelland, T., Paul, NR, Fort, L., Nikolaou, S., Ismail, S. 和 Machesky, LM (2021)。 CYRI-A 通過大胞質體形成和整合素攝取調節來限制侵入性遷移。 細胞生物學雜誌220(9)。




競爭利益_


作者聲明沒有競爭利益。




參考


Bloomfield, G. 和 Kay, RR (2016)。巨胞飲作用的使用和濫用。 細胞科學雜誌 129(14):2697-2705。
Commisso, C.、Flinn, RJ 和 Bar-Sagi, D. (2014)。通過基於圖像的定量測定確定細胞的巨胞飲指數。 國家協議9(1):182-192。
Davidson, AJ 和 Insall, RH (2011)。基於肌動蛋白的動力:WAVE 監管複雜結構重新打開了舊的 SCAR。 Curr Biol 21(2):R66-68。
Goley, ED 和 Welch, MD (2006)。 ARP2/3 複合物:肌動蛋白成核劑成熟。 Nat Rev Mol Cell Biol 7(10):713-726。
Koivusalo, M.、Welch, C.、Hayashi, H.、Scott, CC、Kim, M.、Alexander, T.、Touret, N.、Hahn, KM 和 Grinstein, S. (2010)。 Amiloride 通過降低膜下 pH 值和阻止 Rac1 和 Cdc42 信號傳導來抑制巨胞飲作用。 細胞生物學雜誌 188(4):547-563。
Krause, M. 和 Gautreau, A. (2014)。引導細胞遷移:lamellipodium 動力學和定向持久性的調節。 Nat Rev Mol 細胞生物學15(9):577-590。
Le, AH, Yelland, T., Paul, NR, Fort, L., Nikolaou, S., Ismail, S. 和 Machesky, LM (2021)。 CYRI-A 通過大胞質體形成和整合素攝取調節來限制侵入性遷移。 細胞生物學雜誌220(9): e202012114。
Machacek, M.、Hodgson, L.、Welch, C.、Elliott, H.、Pertz, O.、Nalbant, P.、Abell, A.、Johnson, GL、Hahn, KM 和 Danuser, G. (2009) .細胞突起過程中 Rho GTPase 活性的協調。 自然461(7260):99-103。
Schindelin, J.、Arganda-Carreras, I.、Frise, E.、Kaynig, V.、Longair, M.、Pietzsch, T.、Preibisch, S.、Rueden, C.、Saalfeld, S.、Schmid, B ., et al ., A. (2012)。斐濟:一個用於生物圖像分析的開源平台。 Nat 方法9(7):676-682。
Swanson, JA 和 Watts, C. (1995)。巨胞飲作用。 趨勢細胞生物學5(11):424-428。
Theveneau, E. 和 Mayor, R. (2013)。上皮和間充質細胞的集體細胞遷移。 細胞分子生命科學70(19):3481-3492。


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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2022 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Le, A. H. and Machesky, L. M. (2022). Image-based Quantification of Macropinocytosis Using Dextran Uptake into Cultured Cells. Bio-protocol 12(7): e4367. DOI: 10.21769/BioProtoc.4367.
  2. Le, A. H., Yelland, T., Paul, N. R., Fort, L., Nikolaou, S., Ismail, S. and Machesky, L. M. (2021). CYRI-A limits invasive migration through macropinosome formation and integrin uptake regulation. J Cell Biol 220(9): e202012114.
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