TITLE: Quantifying Fluorescent Cells in Mammalian Cell Tissue Culture
AUTHORS: Harley King

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Quantifying the change in fluorescent status of cells over time can be an important data point in experimental analysis.  Optimally, quantification can be accomplished with flow cytometers. However, these instruments are not available to every lab. Routine analysis and multi-sample preparation can be both time and cost prohibitive.</div><div class = "text-block">Fluorescent cell quantifcation in cell culture requires a fluorescent microscope and freely available imaging software like Image J. </div></div>

[STEPS]
?. [Preparation and Transfection of Cells]
A six-well plate is seeded with cells at a typical pre-transfection density. The following day cells are transfected.
?. [Capture Plate Images]
Images can be taken of the plate 24 twenty-four hours post transfection (hpt). Depending on the vector and the promoter, fluorescence may not be observed. Images of the plate can also be taken at 48 and 72 hpt.Save images in a folder renamed to include the hours after transfection, e.g. "6-w.plate.images.48hpt".
The LSM 710 confocal microscope requires a plate adapter. With this plate adapter, only the 10x objective can be used. Experimenters wishing for greater magnification may need to use chamber slides.
Take a series of adjacent images in the plate's center to minimize shadows cast by plate walls. Images should not overlap. Fluorescence and brightfield should be overlayed. Additional excitatory channels may be added to verify fluorescent vs. autofluorescent emissions.
?. [Batch Process Images Using Zen Lite, Photoshop and Image J]
Quantifying fluorescent cells in images is complicated. The gold standard is eye-hand counting, or couting every fluorescent object in an image with a counter. However, multiple images from six wells at three different time points can result in hours of fluorescent cell counting.Current software packages allow for algorithmic counting of fluorescent cells. CellProfiler allows for image-manipulation pipelines and batch-mode processing but can be difficult to setup. ImageJ (Fiji implementation) is easier to setup but is more limited in automation and cellular recognition.This tutorial uses Fiji.
?. [Batch Process Images Using Zen Lite, Photoshop and Image J]
Before importing the files into Image J, they must be adjusted so that the fluorescent layer is as bright as possible. Do this for all images systematically i.e. use the same values across all images to standardize them.Use Image J or confocal-specific software to isolate the fluorescent channel. In this protocol, images are processed with Zen Lite (v 2.6) using GFP fluorescence. Using Zen Lite, in "Camera" mode, each file is opened. The green channel histogram is set from default 255 to 50. The image is saved, then closed. Modified images were saved as in their binary format (.czi) in the same folder, different than the parent folder.Next, "Processing"-->"Bacth" mode is selected. Select all .czi files in the folder and export using the following settings:file type: "Tagged Image File Format (TIFF)"checked "Convert to 8 bit"Compression "None"Resize: 100%"Apply Display Settings and Channel Color"Checked: "Individual Channels Image"Selected: "Define Subset". Channels: "Ch2-T1 deselected, T PMT-T1 deselected"; "Ch1-T2 selected". Shows as "3".The "Copy Parameters" button was selected. All files were selected and then "Paste Parameters". Added files were i) checked to ensure the same Tif export parameters were duplicated for each individual file.
?. [Batch Process Images Using Zen Lite, Photoshop and Image J]
Exported .tiff images are opened in Adobe Photoshop to covert images to black and white and to invert the colors. "Image"-->"Adjustments"-->"Black and White". Set green to "300%"."Image"-->"Mode"-->"Grayscale". Select "Flatten" and then "Discard" to prompt requests for warnings about image flattening and layer loss."Image"-->"Adjustments"-->"Invert"Export as a .tiff file.
Scripts or "Actions" can be written in Photoshop and then executed en batch.
?. [Batch Process Images Using Zen Lite, Photoshop and Image J]
Get Fluorescent Cell Counts in Image J by importing the image. Select "Analyze"-->"Analyze Particles". Adjust the size and circularity settings so that a majority of the fluorescent cells are properly labeled after selecting "OK". Ensure that counts from the images are properly recorded in a text file or spreadsheet. Image J will prompt for saving.
Image J settings can be saved to a "Macro" which can also be used for a batch process.
?. [Analyze Results]
Analysis software like GraphPad Prism or Excel can be used to view the results.   ABCDEFGHIJKLMNOPQRSTUVWXY1TIMEWell1_1Well1_2Well1_3Well1_4Well2_1Well2_2Well2_3Well2_4Well3_1Well3_2Well3_3Well3_4Well4_1Well4_2Well4_3Well4_4Well5_1Well5_2Well5_3Well5_4Well6_1Well6_2Well6_3Well6_4224hpi140122152147195230234235209193200242113132124122213247220201199183156221348hpi11411515816796123163150250256261225134126151119363371352343263269284243472hpi15313010712814191612981107888139134135137247274244227368437385297
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