Isolamento e caracterização de células dendríticas e macrófagos a partir do intestino do rato

The overall goal of this procedure is to rapidly isolate mouse intestinal dendritic cells or dcs and macrophages for phenotypic and functional characterization. This is accomplished by first harvesting intestinal tissue. The second step is to dissociate the intestinal epithelium from the Lamin propria.

Next, the intestinal Lamin Propria is digested during the final steps of the procedure. The Lamin propria cells are stained with fluorescence, conjugated antibodies, and gated for specific cell populations. Ultimately, intestinal DC and macrophage populations can be more accurately characterized and purified for functional studies to assess cytokine production antigen presentation and the regulation of immune cells.

So the main advantage of this technique over existing methods is that the rapidity of the tissue digestion ensures optimal cell surface antigen expression, cell viability, and or cellular. After euthanizing the mouse and confirming death by the toe pinch reflex, test spray 70%ethanol onto the abdomen and thorax of the animal. Then use a pair of scissors to make a small horizontal incision in the middle of the abdomen and peel back the skin to expose the peritoneum.

Open the peritoneum using scissors after cutting at the pilo sphincter to separate the stomach from the upper small intestine and using forceps to tease away the mesentery. Cut again at the ileocecal valve to free the entire small intestine from the large intestine. Continue to tease apart the mesentery from the large intestine and make a cut at the anal verge.

Use scissors to cut the colon open longitudinally and use room temperature C-M-F-P-B-S to wash the fecal contents and mucus out of the intestinal lumen. Then use the scissors and forceps to macroscopically. Dissect out the pyres patches along the anti mesenteric surface of the small intestine, and cut open the small intestine longitudinally.

Next, clear the small intestine lumen of fecal contents and mucus with more room temperature C-M-F-P-B-S. Separately, cut the small and large intestines into approximately 1.5 centimeter pieces and place the pieces into individual 50 milliliter conical tubes containing 30 milliliters of pre-war C-M-F-H-B-S-S with FBS and two millimolar EDTA. Place each 50 milliliter conical tube horizontally into an orbital shaker shaking the tubes at 250 RPM for 20 minutes at 37 degrees Celsius.

Next place a single mesh wire strainer over a waste bucket. Pour the contents of each 50 milliliter conical tube through the mesh to recover the pieces of intestine and then place them in new individual 50 milliliter conical tubes containing 30 milliliters of Prewarm C-M-F-H-B-S-S with FBS with two millimolar EDTA. Finally, place the tubes back into the orbital shaker for 20 more minutes.

After the second round of shaking, pour the contents of each tube through the strainer. Again, dab away the excess media using a paper towel, and then transfer the pieces of intestine to a small plastic whey boat. The single most difficult aspect of this technique is the tissue digestion to ensure success, all the different parameters of the duration of digestion, the properties of the collagenase, the tissue integrity and the size of the tissue to be digested must all be considered Using scissors rapidly.

Chop the intestinal pieces and then add the minced intestine into a 50 millimeter conical tube containing 20 milliliters of collagenase solution. Place the conical tube horizontally into the orbital shaker and digest the pieces of intestine at 200 RPM for 10 to 20 minutes at 37 degrees Celsius. Now briefly vortex the tube to ensure thorough dissociation of any remaining intestinal tissue and then filter the cell slurry through a 100 micron cell strainer directly into a 50 milliliter conical tube.

Finally, top off the conical tube with C-M-F-H-B-S-S with FBS and centrifuge the cell solution two times for five minutes at 425 times G at four degrees Celsius. Then pour off the supernatant and resuspend the cell pellet in ice cold C-M-F-H-B-S-S with FBS and place the samples on ice. After staining the cells for the surface proteins of interest.

Begin by creating a dot plot excluding the cells that are positive for the dead cell stain as demonstrated here. Next, exclude doublet events as shown here and here. Now use the forward and side scatter channels to gate on the cells of interest, making sure to exclude debris.

Then create another dot plot gating on the CD 45 positive and IAB positive antigen. Presenting cells in a separate dot plot create regions for distinguishing specific DC and macrophage subsets. The R one region shown here captures the CD 11 C positive CD 11 B dull negative cells.

The R two region delineates the cells that have a similar level of surface expression for CD 11 C as the cells in the R one region, but that also express CD 11 B.The R three regions designate the cells that are CD 11, B positive, and CD 11 C dull negative. These three regions can be further analyzed for F four 80 and CD 1 0 3 expression to differentiate macrophage and DC populations respectively. As seen in this dot plot, the CD 11 C positive CD 11 B dull negative cells in the R one region will express high levels of the alpha ein CD 1 0 3 and low levels of F four 80.

This dot plot shows that CD 11 B positive CD 11 C positive cells in the R two region will be composed of both DCS and macrophages based on their dichotomous expression of CD 1 0 3 and F four 80. Finally, based on their F four 80 positive and CD 1 0 3 negative phenotypic profile R three, gated CD 11 B positive CD 11 C del negative cells will consist of macrophages, the relationship between the duration of tissue digestion on total cell yield and the expression of CD 11 B and CD 11 C is illustrated in the following six figures. Intestinal tissue that is digested for three minutes yields a low total cell number resulting in few dcs and macrophages available for characterization tissue digestion for 11 minutes produces a robust yield of live cells, yielding populations of dcs and macrophages that express high levels of CD 11 B and CD 11 C that are phenotypically distinct.

In contrast, digestion for 50 minutes results in a similar cell yield compared to optimized digestion. However, delineation of different cell populations using CD 11 B and CD 11 C becomes more difficult. As the expression of CD 11 C diminishes and the number of dead cells increases.

So upon optimizing the parameters for intestinal tissue digestion, a robust cell yield can be rapidly achieved. As a result, the rapid isolation of mouse intestinal dcs and macrophages can be useful for evaluating the functional properties of immune cells with intact surface antigens.