Workflow for High-content, Individual Cell Quantification of Fluorescent Markers from Universal Microscope Data, Supported by Open Source Software

The overall goal of this procedure is to objectively measure the responses of individual cells by quantifying specific fluorescent marker intensities from microscope images. This is accomplished by first subjecting a adherent tissue culture cells to irna mediated gene knockdown. In the second step, the cells are fluorescently stained and then imaged for several markers of interest.

In the final step, the expression of the markers within specific subcellular regions are algorithmically defined, are individual cells. Ultimately, the cellular responses to biological cues from gene knockdown can be analyzed by measuring the fluorescent expression levels of the markers of interest and their subcellular Translocation. Though this procedure provides insight into the regulation of G one cell cycle transit in response to siRNAs.

It can also be applied to studies generating fluorescent cell images, studying nuclear transport and protein homeostasis. Demonstrating the procedure will be Daniel Wek and Car K HO fellow postdocs from my laboratory Begin by dispensing 70 microliters of 200 nanomolar irna, diluted an irna buffer into the appropriate wells of a sterile 96 well plate in a sterile tissue culture hood. Then add 105 microliters of transfection lipid diluted in 40 volumes of serum free DMEM medium into each well of sir a mix the plate by gentle vibration of room temperature, and then after 10 minutes, subdivide the resulting 175 microliters into three 50 microliter replicates per target into an opaque tissue culture treated 96 well plate with a transparent base.

Next, dispense 8, 000 cells per well in 150 microliters of DMEM with 10%serum directly onto the 50 microliter lipid irna complexes without mixing. Then seal the plate with a sterile adhesive breathable membrane and place the plate into a humidified incubator at 37 degrees Celsius and 5%carbon dioxide. 48 hours later aspirate the media from the plates.

Then fix the cells in 100 microliters of 4%buffer formaldehyde in a fume hood at room temperature After 10 minutes, aspirate the fixative and perform three 100 microliter washes with PBS after the last wash. Remove the PBS and perforate the cells with three cycles of 100 microliter perme ization solution incubations of 10 minutes each at room temperature without shaking. After the last incubation, remove the permeation solution and then add 100 microliters of block solution per well for 30 minutes at room temperature.

Then aspirate the block solution and probe the cells with 50 microliters of the primary antibody of interest within two weeks of labeling uc confocal microscope with a 20 x objective to take separate 16 bit gray scale TIFF images in three channels corresponding to the DNA dye GFP and immuno staining. Flora fours capture many non-overlapping image sets or frames to image approximately 1000 to 2000 cells per well. Naming the image file systematically with the metadata information so that each file name is a unique combination of experiment name, well address, frame number, and channel identifier.

Then open the cell profiler software and click file and import pipeline. Next, under from file, select the file three channels pipeline CP pipe file, which contains the necessary instructions for the software to interpret the image file metadata from the saved file name Convention Cell Profiler can now be used to relate the images, extract the nuclear DNA and antibody intensities from the files, and use the GFP channel to calculate the ratio of the nuclear versus cytoplasm intensity. For each cell detected, click the view output settings button and then click the default input and default output folders.

Selecting the location of the image files for analysis and the destination for the extracted data respectively. Then in the analysis modules window, click the appropriate eye icons to observe the identify primary objects and tertiary objects windows during the analysis and click analyze images when the analysis is complete. Click okay in the message box and go to the default output folder Location where all the data files are saved as comma separated value files.

To perform the data extraction, find the new nuclei CSV file included among the output from cell profiler, which contains the individual cell data for the fluorescent nuclear antibody intensity nuclear DNA intensity and GFP CDK two reporter ratio values. Copy the provided pulse script file to gait classifier PL into the same folder as the nuclei CSV data file. Then double click the icon for Pearl Script.

And when prompted, type the full name of the data file followed by a DOT CSV file name for the file in which the cells are to be gated and the gate values for the antibody fluorescence and G FP CDK two reporter data. Confirm that the newly created file combines the raw individual cell assay values from the original cell profile of data with the subpopulation labels showing how each cell from each well performs against both gates. Now open the R Studio software to plot the data for each experimental condition using the individual cell subpopulation labels by clicking file and open file, and then selecting the provided analysis dot r file.

Type the computer address of the folder containing the gated data, including the drive letter and the name of the file itself. Then highlight lines one through 17 in the upper left window of our studio, and click on the run button to enter the experimental data threshold values and well location details. Finally, highlight the individual blocks of the remaining code beneath line 17, and click the run button to create the corresponding plots.

Observe the plots in the window in the lower right corner of our studio, and then click the export button to save these in a variety of formats. The workflow described here analyzes the fluorescent microscope images to produce raw data in the form of a list detailing each cell identified and its corresponding assay values. Many options exist for the analysis of these data.

For example, in these histogram plots, the assay values for the cells treated with control RNAs enable the identification of the appropriate gates to threshold each assay. The gating thresholds are then applied using a pulse script to label each cell in the raw data according to the assay outcome. This labeling approach assists the preliminary visual as well as the automated assessment of the relationships between the treatments and the subpopulation distribution of even very large sets of individual cell data.

For example, in this representative experiment, threes irna knockdown conditions resulted in contrasting distributions of the cells relative to the gates are the two assays. The R plots use the labels to calculate the percent distributions of the cells in each quadrant. The labels also enable additional fluorescent measurements to be cross-referenced with the assay data.

In this experiment, the subpopulations of cells treated with SI CDK six were grouped according to their respective assay labels and plotted as histograms of nuclear DNA staining. This use of the cell data labels reveals the relationship between the two cell assays and the DNA content in the cells. When performing this procedure, it is always important to remember to test and adjust the image segmentation parameters when using cell profiler.

This is particularly important when using new or untested image files for the first time.Okay.