Proteomic Profiling of Macrophages by 2D Electrophoresis

The overall goal of this procedure is to analyze the phenotype of human pro-inflammatory M1 and anti-inflammatory M two macrophage subsets by 2D differential in gel electrophoresis. This is accomplished by first labeling proteins extracted from primary M1 and M two macrophage cultures with sine dyes. In the second step, the proteins are mixed together and ISO electro focusing by rehydration is carried out on the samples.

Next second dimension, electrophoresis is performed with a mobilized pH gradient or IPG strips in the dark, and then the gels are scanned with the differential in gel electrophoresis scanner. Ultimately, analysis of the numerated images is performed to detect the differences in the polyp peptic spots between M1 and M two macrophage subsets. The main advantage of this technique among existing methods like classical todi gel electrophoresis, is that this technique is more sensitive, requiring only five microgram of proteins, which is very important for samples obtained from human patients.

Demonstrating the procedure will be Mario Bove, a PhD student, Olivia Beza and Magac technician from my laboratory To prepare primary cultures of monocyte derived macrophages begin by diluting 25 milliliters of Buffy coat in 25 milliliters of PBS. Then carefully load the diluted blood onto a separation gradient and centrifuge the cells for 20 minutes at 1600 Gs and room temperature when the cells have finished spinning. Collect the monocytes at the interface layer, followed by three successive washes of the harvested cells in 10 milliliters of PBS containing 0.1%EDTA after the third wash.

Spin the cells down one more time in PBS alone, and then resuspend the pellet in five milliliters of RPMI 1640 medium without serum seed. The cells in 35 millimeter dishes at a density of one times 10 to the six cells per dish, and then after a 90 minute sedimentation in the incubator, discard the supernatant and wash the adherent monocytes three times with one milliliter of PBS. Next culture, the cells in 10 milliliters of fresh medium containing 10%volume per volume of human serum for six days at 37 degrees Celsius and 5%carbon dioxide.

Then to yield anti-inflammatory M two macrophages, supplement some of the cultures with 15 nanograms per milliliter of IL four on day six of culture to yield pro-inflammatory macrophages on day 12. Treat the other cultures of differentiated macrophages with 100 nanograms per milliliter of LPS for four hours to isolate the macrophage subsets for 2D electrophoresis. Next, wash the culture of interest three times with 25 millimolar tris, and then release the cells from the bottoms of the plates with chaps extraction buffer.

Next, transfer the cells into a 1.5 milliliter micro centrifuge tube and lace them at four degrees Celsius. After five minutes, transfer the cells in 100 microliter aliquots to minus 20 degrees Celsius storage until the protein concentration analysis for ISO electro focusing of the macrophage subsets. First, adjust the samples to nine microliter aliquots, and then reduce the cell solutions with lysis buffer supplemented with two millimolar of TCEP at 37 degrees Celsius.

After one hour, incubate the extracts with S SI three and S SI five at 37 degrees Celsius, stopping the reaction after 30 minutes with the addition of an equal volume of sample buffer. Then create two separate mixtures of equal volumes of S SI three labeled M1 proteins with SCI five labeled M two proteins. Next rehydrate an IPG strip with 450 microliters of the labeled mixed samples in chaps extraction buffer on an isoelectric focusing cell system for 24 hours without applying any current.

The next day, initiate the focusing at 300 volts for three hours, then set up a gradient to 1000 volts for six hours, followed by two 8, 000 volt gradients for three hours each to perform second dimension electrophoresis incubate the IPG strips in equilibration buffer. After 10 minutes, transfer the strips onto 12.5%page gels and seal them with low melting aros. Then use an edin dsic system at a constant voltage of 70 volts to carry out the electrophoresis at 20 degrees Celsius overnight, followed by a 300 volt run until the bromo phenol blue front reaches the bottom of the gel to acquire images of the gels.

Place them between the two low fluorescence glass plates of a differential and gel electrophoresis imager scanner, and scan the gels at excitation emission wavelengths of 5 32, 5 80 nanometers fors three and 6 33 6 70 nanometers fors five to yield images with a pixel size of 100 micrometer. Analyze the with the appropriate software and then calculate and normalize the spot volumes in each image, assigning a normalized spot volume as a proportion of the total value of each spot detected in the gel. Analyze the differences in protein spot volumes for each type of macrophage by comparing the normalized spot volume value between the two macrophage groups, considering the difference between the spot volumes to be significant if the change is 1.5 fold.

In this experiment. The 2D protein patterns of the two subtypes of macrophages M1 pro-inflammatory and M two anti-inflammatory were determined by 2D differential. In gel electrophoresis, the same pattern of proteins was observed for either M1 or M two independently of the cyan dyes used.

Interestingly, bioinformatic analysis of the gel revealed 20 areas containing spots, increases and decreases between the spot volumes were quantified as visualized by the yellow, blue, and green coloring spots were considered to have a significant differential expression if the fold change of the normalized spot volume was greater than 1.5 with a P value less than 0.05. Following this CHE, offer methods like quantitative, mass spectrometry or additional proteomic analysis can be performed to answer additional questions such as what type of proteins are differentially expressed between the two macrophage subsets in response to various stimuli.