Overview

After performing an experiment involving the Mef subculturing 24-well plate protocol or similar, the wells must be imaged and the proliferation data harvested and processed in RStudio. This protocol will outline how to use an EVOS FL Auto to image cell culture well plates that have been stained with a Hoescht blue stain, as well as how to harvest and process relevant cell proliferation data from those images in RStudio.

Protocol

Imaging

  1. Take the well plate to be imaged to the EVOS. Remove the magnetic lid from the top, remove any slide holders from the interior, and insert the well plate such that the plastic tip at the upper left corner is bent back. Place the magnetic lid back on the machine.
  2. Power on the machine and open the EVOS program on the attached computer. Turn attention to the program.
  3. Toggle the Power option to “On.”
  4. Select the Vessel Expert and change the vessel to your preferred well plate type. This protocol is specific to 96-well plates.
  5. Ensure the zoom option is set to “10x” and the filter option is set to only “DAPI.”
  6. Select one of your wells containing cells on the well plate diagram shown.
  7. Adjust the Light slider and Coarse Focus slider until cells are distinguishable. Select “Capture Focus Nominal.”
  8. Adjust the Fine Focus slider (or use Auto-Fine operation) until cells are crisp and in focus. Select “Capture Focus Nominal” again.
  9. Move to the “Scan” tab and create a new scan in an appropriate folder (or edit an existing one, if applicable).
  10. Select 10x zoom, monochrome, and DAPI. Move to next page.
  11. Select “select wells.” Move to next page.
  12. Select the desired wells, “serpentine” path, and check “show this page again” at the bottom. Move to next page.
  13. Select “center of each well” and “DAPI” for both Find Focus and Auto Fine options. Move to next page.
  14. Select “center region only” and input 7% for how much of the center region to scan (it should be taking 6 images per well). Move to next page.
  15. Find your cells using the methods in Steps 6 through 8. There is no Capture Focus Nominal option here, so ignore that. Check “show this page again” at the bottom. Move to next page.
  16. Save and run the scan.
  17. Save your images, power down the EVOS machine, take your well plate, and make sure to put back the magnetic lid and sign in and out on the sheet.
If an image is taken out of focus
  1. Cancel the scan and save images taken up to that point.
  2. Return to the initial “Image” tab and find your cells as shown in Steps 6 through 8. Make sure to Capture Focus Nominal.
  3. Run the scan again, this time selecting only the wells that have not yet been properly imaged (including the one that was blurry before).
  4. Repeat as necessary until all wells are crisply imaged.

Image Processing in ImageJ

  1. Open well image in ImageJ.
  2. Run the following macro:

for (i=0; i<1;i++) { 

run("8-bit");
setAutoThreshold("Default");
//run("Color Threshold...");
setThreshold(17, 40);
//setThreshold(17, 40);
run("Convert to Mask");
run("Convert to Mask");
run("Watershed");
run("Analyze Particles...", "size=30-1000 show=[Overlay Masks] summarize overlay");
saveAs("Tiff", "/Users/YourName/Library/Mobile Documents/com~apple~CloudDocs/YourWorkFolder/PROJECTS/YourProject/todays-date-00-00-00/0placeholder.tif");
run("Open Next");

}

  1. Repeat the macro for each image on a well plate, modifying thresholds as necessary. If you're feeling confident, change the index to something greater than 1. If you don't know what the index is or how to change it, you shouldn't be feeling this confident yet.
  2. When finished processing images for a well plate, delete any duplicate entries and save the summary as a CSV.
  3. Repeat as necessary for all well plates.

Data Processing in RStudio