Howtoperformautomatedcountsoffluorescentlystainedcells.

Note
This protocol describes semi-automated cell counts using fluorescently labeled cells, a hemocytometer and ImageJ software.  The hemocytometer is not needed if you have already calibrated the areas of images taken with your microscope and camera.  Our setup automatically imports images in iPhoto and loads them into Photoshop with a double-click.  However, you could also import and crop the images directly into ImageJ if you prefer.

Materials
Cells in media
EtOH
1x P.I. (100 µg propidium iodide / mL PBS)
Hemocytometer
U.V. fluorescent microscope with camera
Computer with ImageJ and image cropping software.

Procedure
Fix Cells by diluting them 50% in EtOH.
1.  To an eppendorf add 0.1 mL cells in PBS or media
2.  Add 0.1 mL of 100% EtOH while vortexing.
3.  Spin at 3,000 RPM x1 min.
4.  Aspirate off supernatant and respend cells in 0.1 mL of 1x P.I.  Mix, incubate 2 min. 

Photograph cells on u.v. microscope
5.  Add 10 µL of stained cells to Hemocytometer.
6.  Place on U.V. microscope. (If the cells are too crowded then you may need to further dilute them in P.I. or PBS)
7.  Photograph 1 large square of hemocytometer with brightfield.
8.  Without moving the stage or changing the zoom, photograph the same area under u.v. light with red filter.  

Import and crop photo (e.g. using iPhoto and Photoshop)
9.  Import photos from the camera to the computer (e.g. with iPhoto).  Double click an image to open image in Photoshop.
10. In Photoshop, use the marquee tool measure the size of large square of the brightfield hemocytometer image.

(A large square on the hemocytometer is 1mm x 1mm x 0.1mm = 0.1 µL)

11. Crop the u.v. fluorescent image of cells to same size as a large square.
12. Convert to grayscale (Image > Mode > Grayscale).
13. Save file as .jpg image (medium resolution).

Count cells in cropped image using ImageJ
14. Open .jpg file with ImageJ software.
15. Convert to binary image (Process > Binary > Threshold)
16. Count cells (Analyze > Analyzed Particles... check Display results, Clear results table, Summarize).
17. Repeat counts with additional large squares to be sure that the cells are evenly distributed and to minimize stochastic error.  (The standard deviation is ~ sqrt(mean)).  

Note: An alternative to cropping the image is to instead photograph the fluorescent cells with a low level of brightfield illuminescense in order to visualize the lines of the hemocytometer together with the fluorescent cells.  Select the area of the large square for cell counts directly in ImageJ. 


Calculate the cell density
Cell density (cells/mL) = Average Cell Count (cells/large square)  *  10,000 (large squares/mL) * d.f. (dilution factor, if any)