The overall goal of the following experiment is to demonstrate the use of image stream technology to quantify cellular mechanisms of primary immune cells from well-defined patient cohorts. This is achieved by first isolating peripheral blood mononuclear cells or pbmc from blood samples acquired from patients or healthy volunteers. After isolation, the cells are activated and then fluorescently labeled.
Next, the cells are hydrodynamically focused, excited with laser light and imaged on a time delay integration. CCD camera. Finally, images are required using the image stream cytometer and quantification of NF kappa b.
Translocation in each cellular image is performed using the ideas software. Ultimately, the differences between each subject group can be determined through analysis of dot plots, histograms and cell images. The advantage of the image stream is that it combines facts and immunofluorescence.
We're able to look at cell signaling pathways in a lineage specific manner, either single cell or population, and starting with only a small sample of blood. This method can help answer key questions in knowledge by localizing and quantifying signaling intermediates. For example, it can be used to investigate action mediated nuclear translocation of transparent factors or to look at the effects of aging on cell biology.
Leader one from my lab will Demonstrate the technique After isolating peripheral blood mononuclear cells obtained from human volunteers. Resus resuspend the recovered p BMCs at three times 10 to the sixth cells per 0.6 milliliters of medium, and then transfer the cell suspension to 1.5 milliliter tubes. Next, stimulate some of the cells with an activating agent such as the TLR eight LA R 8 4 8 shown here, and then incubate all of the cells at 37 degrees Celsius for 10 to 60 minutes in a 5%carbon dioxide incubator.
After the incubation period, pellet the cells for five minutes at 500 times G at room temperature, discard the supernatant, label the cells with a cocktail of surface antibodies specific for monocyte or DC lineages for 20 minutes at four degrees Celsius, including tubes of cells labeled with single color conjugates to be used as compensation controls. Then fix the cells in 4%P-F-A-P-B-S for 10 minutes at room temperature. After centrifuging the cells for 10 minutes at 500 times G at room temperature, resuspend the pbmc in freezing buffer and store the cells at negative 80 degrees Celsius until they will be analyzed on the day of assay.
Thaw the cells quickly in a 37 degrees Celsius water bath, and then after centrifuging to remove the freezing buffer, perme them with 100 microliters of BD perm wash buffer. Then label with anti intracellular signaling component antibodies such as the anti NF kappa BP 65 antibodies shown here for 20 minutes at room temperature. After pelleting, the cells Resus suspend them in 100 microliters of BD perm wash buffer containing goat anti rabbit IgG.
Alexa 6 47 and incubate the pbmc for 20 more minutes at room temperature. Immediately prior to imaging counterstain the cells with a nuclear dye begin by powering up image stream and then launching the inspire software. Next, click on the load default template and then select initialize fluidics.
Then click on the image gallery menu and select all. Now press run set up to start imaging the beads and then select the bright field channel. Once the speed beads are running, click start all under the assist tab to calibrate and test the instrument.
After the ImageStream system has been calibrated, press flush, lock and load to load the brightest sample into the machine. Then set the laser power such that each flora chrome has a maximum pixel value between 104, 000 counts as measured in the dot plots. Then set the cell classification criteria.
Now click run, acquire to collect and save the experiment data file to analyze the data files first, open ideas. Analytical software create compensated image files and then gate on events with a normal nuclear dye intensity and a high nuclear dye aspect ratio. To distinguish single cells in the R one gate from debris or multicellular events, monocytes are found within the gate for CD 14 positive cells, myeloid dendritic cells, which are high for CD 11 C and low for CD four and lin one markers are in the R three gate.
Finally, use ideas software to determine the colocalization of the cytoplasmic transcription factor NF kappa B with the nuclear dye. A high correlation of NF kappa B nuclear dye indicating colocalization of the two markers is reflected in a high similarity score and indicates the degree of activation of the cell. The percentage of high similarity events between different treatment groups or patient populations can be compared to evaluate the relative functional efficiency of the cells idea.
Software can then be used to display images of cells from key segments of population histograms. ImageStream allows gating of cell subsets directly from pbmc and imaging of cell responses from gated, but unsorted cell populations. Here the effect of TLR signaling on nuclear localization of NF kappa BP 65 in lin one, negative CD four, dim CD 11 C positive myeloid dcs was quantified at baseline.
A high percentage of unstimulated cells with the transcription factor NF kappa BP 65 in the cytoplasm was observed After stimulation, the treated cells were determined to have a significantly higher similarity score of NF Kappa BP 65 with nuclear stain PI indicating that NF Kappa BP 65 trans located into the nucleus after stimulation. Digital images collected simultaneously of the untreated, or R 8 48 stimulated cell populations show representative cells and the intensity of NF kappa B trans located into the cell nucleus. Note the brighter intensity of NF Kappa B and PI staining in the R 8 48 stimulated group compared to the untreated cells.
We've used this technique on cells from different age donors and have shown differences in TLR mediated NF kappa B activation pathways Once mastered, this technique can be done within one day within additional time for image analysis, allowing both several localization image and population statistics. In addition, we can image responses directly from multiple cell population without physical separation. Image stream may be broadly applied to a number of cellular mechanisms and should be very valuable in translational research.
