Rapid and Robust Analysis of Cellular and Molecular Polarization Induced by Chemokine Signaling

The goal of the following procedure is to rapidly and automatically quantify the changes in cell morphology and the subcellular localization of specific proteins of interest in large numbers of cells in response to a particular stimulus. This is accomplished by first stimulating the T cells with the chemo kinds of interest. In the second step, the cells are fluorescently labeled and then analyzed by imaging flow cytometry.

In the final step, the number of morphologically polarized lymphocytes and the amount of segregation of the fluorescent markers can be evaluated based on the location and the intensity of the pixels for each staining, depending on the chosen mask. Ultimately, this imaging flow cytometry technology can be used to rapidly and robustly quantify the morphological changes of lymphocytes after chemokine stimulation, as well as the location of specific proteins of interest within the polarized cells. The main advantage of this technique over existing methods like microscopy is that a large number of cells can be repeatedly processed and automatically analyzed securing statistically strong analysis of the morphological changes and the protein distribution.

In chemokine stimulated T lymphocytes, Boche kind stimulation of the T cells begin by washing five times center the five cells per experimental condition in warm HBSS supplemented with 10 millimolar HEPs centrifuge for five minutes at 340 times G.At room temperature, remove the supinate and re suspend the pellets in 0.3 milliliters of warm HBSS he piece per tube. Then transfer the cell suspensions into individual 1.5 milliliter micro centrifuge tubes. Add 10 to 500 nanograms per milliliter of chemokine to each tube, and then flip the tubes several times to mix.

Incubate the cells in a 37 degree Celsius water bath. After eight to 10 minutes, stop the polarization with 0.3 milliliters of warm, 2%paraldehyde supplemented with 10 millimolar. He peas.

Then mix the tubes with more flipping and incubate them for another five minutes at 37 degrees Celsius to stain the cells for immunofluorescence analysis. Next, transfer the stimulated T cells into corresponding flow cytometry tubes. Fill the tubes completely with staining buffer.

Then spin down the cells and resuspend the pellets in 100 microliters of 5%fetal calf serum in PBS. Now add five microliters of ZI N-T-H-L-A-A-B-C to each tube. Vortex them and incubate the cells for 30 minutes of room temperature in the dark at the end of the incubation.

Wash the cells twice and then incubate them in 500 microliters of 1%para formaldehyde. After 10 minutes, wash the cells in more staining buffer, and then fill the tubes completely with permeation buffer. After washing the cells twice in the permeation buffer left the tubes to remove the supinate and then vortex them to dissociate the cell pellets.

Now stain the samples with zero point 25 micrograms per milliliter, trixi for loin, vortex them and incubate for 30 minutes at room temperature in the dark. Then at the end of the incubation period, wash the cells in permeation buffer. Again, resus.

Suspend the pellets in 200 microliters of PBS and then transfer the cells into individual micro centrifuge tubes to determine the percentage of polarized T cells after chemokine stimulation. First switch on the imaging flow cytometer and let it calibrate itself according to the manufacturer's instructions. Draw histograms that quantify the RMS gradient values from the in-focus population selected on the RMS gradient.

Histogram delineate the single cell population on a histogram of the area once the micro centrifuge tube has been inserted in the machine, adjust the laser power gait on individual cells and acquire 5, 000 T lymphocytes. In the ideas analysis software. Open the acquired files, then create a histogram showing the gradient RMS value for the brightfield images obtained for each individual cell, drawing a gate on the cells, exhibiting an RMS gradient value above 57.

In the analysis menu, create a new mask called erode M1 two from the morphology mask on the brightfield images M zero one eroded of two pixels. Then in the analysis features menu, create a new feature of the area calculated on the erode M1 two mask from the cells created in the previous gate. Open a histogram showing the area of cells Using this newly created mask, draw a gate on the individual T cells, excluding the calibration beads and debris in the small area, as well as the doublets in the large area.

Then open a. bot consisting of the raw max on the H-L-A-A-B-C staining as a function of the raw max pixel on the Phin staining in the analysis masks menu rear to new mask called erode M two two from the morphology mask of the H-L-A-A-B-C stain cells eroded of two pixels. Then in the analysis features menu create a new feature consisting of the circularity parameter calculated on the erode M two two.

Subsequently create another histogram to report the values of the circularity feature from the previously gated cells. Then looking at the shape of the histogram for the non stimulated cells, RO agate for the polarized cells, starting at the lowest circularity value up to the circularity limit value where most of the non stimulated cells are plotted to assess the polarization of the actin staining within the cells. Plus a histogram for the bright detail similarity, R three value to compare the H-L-A-A-B-C and the foid in staining.

Then using the same gating strategy as just demonstrated, plus a GA on the non stimulated cells for the non co localized segregated staining. The statistics panel shows the percentage of T cells with polarized actin staining here. Examples of CCL 19 stimulated T lymphocytes that fall in the gate of the non-polarized or polarized cells are shown.

Differences in the morphology between both subpopulations can be clearly observed. The just demonstrated gating strategy can also be used to plot the percentage of cells that fall in the polarized gate in the absence of presence of CCL 19. As expected, it's clear that chemokine stimulation increases the percentage of polarized T cells.

The mean circularity index of the whole cell population obtained under both conditions can be plotted as well. Indeed, CCL 19 stimulation elicits a drop in this index. However, as only half the cells are polarized, the change in the mean circularity index observed upon CCL 19 stimulation is small.

In this experiment, the T cells were cot transected with GFP together with plasmids encoding for the dominant negative mutants of the polarity complex par six. PKC zeta. The histogram for the nont transfected cells allowed specific gating for the relevant GFP positive T cells indicating the to small fraction of the whole cell population have been transfected.

The degree of morphological polarization of these transfected cells was then quantified by measuring only the circularity index of the GFP positive cells and an additional gait for the T cells. Exhibiting low values of circularity was created to estimate the percentage of polarized cells under each condition taken. Together these data demonstrate that disruption of the function of the par six PK CZ to signaling pathway inhibits T-cell polarization induced by both CCL 19 and c Xcl 12 chemokines.

In line with previous findings. While attempting this procedure, it is very important to always work at 37 degrees Celsius throughout the insolution kin stimulation step and the following fixation to ensure strong and reliable morphological polarization of the lymphocytes.