Isolation of Leukocytes from the Human Maternal-fetal Interface

The overall goal of this procedure is to isolate leukocytes from the human decidua, baus, and decidua paral. This is accomplished by first dissecting the human dee tissues from the placenta. Next, the decidual tissue is dissociated by mechanical disaggregation and enzymatic digestion.

Then the cell suspension is filtered using a 100 micrometer cell strainer washed with one XPBS and fuge at 300 G for five minutes at four degrees Celsius to obtain a cell pellet. Finally, the cell suspension is loaded on top of the density gradient solution and centrifuge, and the interface containing leukocytes is collected. Ultimately, isolated leukocytes can be used for immunophenotyping cell culture and functional studies.

The brain advantage of this technique or existing methods, such as mechanical dissociation or enzymatic digestion, is that it combines mechanical tissue desegregation with an automatic tissue associator and thematic digestion with Accutane and leukocyte isolation using a fecal gradient. This protocol has been proven to preserve cell surface antigens in cell viability, and its implications extend toward the understanding of the functional and phenotypic properties of infiltrating leukocytes at the maternal fetal interface related to the pathogenesis of prenatal complications. The collection and utilization of human samples for research purposes were approved by the institutional review boards of the Eunice Kennedy Shriver, national Institute of Child Health and Human Development, national Institutes of Health, department of Health and Human Services, and Wayne State University.

Written informed consent was obtained from all pregnant women prior to the collection of tissue samples.Yay. To begin with, the deci basilis dissect a piece of the basal plate from one co leadin of the placenta. Place it on a sterile cutting board with the placental vli, which will be bloody red with a hairy appearance facing upward.

The basal plate is smooth and pale red in color while keeping the tissue soaked in one XPBS use sharp, fine point scissors and forceps to remove the villous tissue and blood vessels. Collect two to three pieces and use PBS to thoroughly rinse them to remove the blood. To isolate the deci paral, dissect the 10 centimeter by 10 centimeter square piece.

Place it on the board with the corion, which contains blood clots and is usually light yellow in color facing upward. Next, use fine point forceps to remove as many of the blood clots as possible. Then with sterile one XPBS, rinse the membrane until the PBS runs clear.

Using a disposable sterile cell scraper and while putting PBS on the membrane, gently scrape off the decidual layer. Collect the deci tissues and place them in a 50 milliliter plastic tube with sterile PBS to mechanically disaggregate the decidua Bali or the deci paral. Begin by using sterile one XPBS to wash the tissue in a 50 milliliter tube.

Then centrifuge the tissue at 300 G and room temperature for five minutes. Carefully aspirate the snat without disturbing the pellet. Then after resus suspending the tissue and detachment solution according to the text protocol, transfer the homogenized tissues to a C tube.

Place the tube in the automatic dis associator and run the corresponding program after the dissociation at a commercially available cell detachment solution, such as Accutane to the tissues, incubate at 37 degrees Celsius with gentle agitation for 45 minutes to digest it. After the incubation, add 10 milliliters of PBS to the digestion mixture and pass it through a 100 micrometer cell strainer into a 50 milliliter tube. Depending on the stickiness, several strainers may be necessary to strain one digestion.

Use PBS to fill the tube and centrifuge at 300 G and room temperature for five minutes. Carefully remove the S supernatant and use five milliliters of ice cold fax buffer to resuspend the cell pellet. To study mononuclear leukocytes, add five milliliters of 20%density gradient medium to a 15 milliliter plastic tube.

Then slowly overlay the cell suspension on top, taking care not to allow the layers to mix with a swing out rotor centrifuge at 500 G at four degrees Celsius and without the break for 30 minutes, leukocytes will be found in the interface between the density gradient medium and the fax buffer. Next, carefully pipette the upper layer that contains the fax buffer and cell debris. The interface contains the residual mononuclear cells and a portion of the polymorphonuclear leukocytes.

Transfer this layer to a new 15 milliliter tube. Add at least three volumes of fax buffer to the tube, then centrifuge at 300 G for five minutes. To palate the cells, aspirate the S supernatant and wash the cells with fax buffer a second time before carrying out cell culture cells.

Sorting and immunophenotyping according to the text protocol, this figure shows a morphology of isolated macrophages collected from the decidua pariahs in a term pregnancy using magnetic cell sorting. Isolated macrophages maintain the ability to release cytokines after three days of culture. The yield of viable cells isolated from the decidua, baus and decidua paral was determined following density gradient separation.

Using a fixable viability as shown here, it was greater than 90%for each deci cell population. Shown here is the gating strategy for analyzing polymorphonuclear and mononuclear leukocytes within the visible gating, including T cells, neutrophils, macrophages, NK cells, NK T cells, and B cells. At term pregnancy, this figure shows neutrophils, macrophages, T cells, and B cells from isolated residual cells from a term pregnancy.

Finally, NK NKT and T cells from isolated residual cells from a term pregnancy are represented in this set of plots. Once mastered, this technique can be done in three hours if it is performed properly. While attempting this procedure, it is important to remember to clean the tissue well, to avoid blood contamination, and to keep the tissue and enzyme cocktail on eyes during the procedure.