Een geoptimaliseerde Procedure voor fluorescentie-geactiveerde celsortering (FACS) Isolatie van autonome neurale voorlopercellen uit viscerale organen van de foetus Muizen

The overall goal of this procedure is to derive a population of purified neuronal progenitors from peripheral nervous system ganglia. This is accomplished by first dissecting the organ or specific ganglia of interest from fetal mice. The tissues are then partially dissociated to free the individual cells into the surrounding media.

Next, the cell suspension is filtered to remove any residual tissue clumps or large aggregates. Finally, the cell suspension is flow sorted to isolate the population of interest based on the expression of a fluorescent reporter in that cell population. Ultimately, gene expression patterns can be compared between progenitors, isolated at different stages of development, or between purified populations of different neuronal cell types.

The main advantage of this technique over isolation of cell populations based on antibody labeling is that often immuno reagents are not available. That label cells expressing a particular gene, genetically engineered lines of mice that produce fluorescent protein in discrete neuronal subsets enable isolation of these cell populations. While we demonstrate this method for isolation of neural progenitors from intestine and neurogen track of the mouth, it can also be applied to other model systems that express fluorescent reporters.

Generally, individuals new to this method will struggle because they tend to over digest the source tissues resulting in a loss of cell viability. Before starting the dissection, view the embryos by brightfield illumination to ensure that the relevant structures are in focus. Then identify the transgene positive embryos by screening for expression of the fluorescent reporter using a fluorescent stereo microscope.

After euthanizing, the embryos begin by adding a minimal amount of PBS to the dissection dish, so the embryo does not float while tissues are dissected. Open the skin of the embryo along the sides of the body and across the abdomen at the level of the liver. Roll the embryo on its back and use fine forceps to pin it in place.

At the forelimb level, insert another pair of forceps at the level of the diaphragm, and then briskly. Pull the internal organs down towards the tail and away from the dorsal body wall to remove the viscera from the liver down to the genital tubercle. If any organs from the thoracic cavity, such as the heart or lungs come out attached to the viscera, remove those before proceeding further.

Once the abdominal viscera are separated from the carcass, insert a pair of forceps below the liver, near the stomach, and gently tease the liver away from the intestine. Then carefully dissect the hind gut away from the dorsal urethra and remove the intestine from the urogenital tract. Finally, turn the intestine over so the spleen is visible beneath the stomach.

Remove the spleen and pancreas. Pull the loops of gut apart by holding onto the mesentary. Then strip off the mesentary and accompanying vasculature from the intestine by gently pulling on the mesentary.

Avoid holding the intestine directly with the forceps. If the intestine breaks, just pool the separate pieces at the dissociation stage. Pool each tissue type together in a 15 milliliter conical tube containing ice cold HBSS according to tissue type and embryo phenotype type.

After pelleting, the sub dissected tissues aspirate as much HBSS as possible. Then resuspend the tissue pellet in one to several milliliters of ACU max. Changing pipette tips between each sample.

To avoid cross contamination of tissue types, place the tubes into a 37 degree Celsius water bath for 20 to 45 minutes. Depending on the stage of the tissue being isolated, Dissociation and filtering of the tissue is the trickiest part of the procedure to prevent overt dissociation of the tissue. And as a result, low cell viability.

Limit the dissociation time and do not try to completely dissociate all of the tissue pieces Halfway through. And at the end of the dissociation time, manually break up the tissue by vigorously knocking the tube against the side of the water bath and flinging the tube down with a snapping wrist motion. Now move the dissociated samples onto ice and immediately add one milliliter of freshly prepared quench to each tube using a new pipette tip for each sample, tri the sample up and down until the tissue is almost completely dissociated.

Now using a pair of ethanol sterilized forceps, place a three centimeter square of 38 micron nylon mesh membrane over the mouth of a new 15 milliliter conical tube. Then use narrow bore tips to filter the individual resuspended cell solution through the mesh by pipetting into the center of the membrane. Once the cell suspension has been filtered, rinse the sample tube with one milliliter of quench one to five and filter any remaining cells.

Then remove the membrane being careful to prevent any unfiltered tissue samples from entering the tube. After pelleting, the cell suspensions aspirate the supernatant and resuspend the pellet in one milliliter of quench one to five. Then filter each cell suspension through a nylon mesh into five milliliter polystyrene tubes as just shown.

Begin by reserving a small aliquot of the fluorescence sample for the EGFP only compensation control. Then divide the wild type tissue sample into two portions labeling one wild type only and the other seven A a d.Only. Now fill all sample tubes with quench one to five, and after pelleting the samples aspirate all but the last 200 microliters of supernatant in each tube.

Finally, add seven a a d to the samples to be sorted and the seven a a d only control. Then add 0.75 milliliters triazole LS to 1.5 milliliter micro centrifuge. Sample collection tubes begin by using the control samples to set the compensation parameters and gates to avoid seven A a D positive dead cells and to collect cells exhibiting a high intensity of GFP fluorescence.

After setting the compensation parameters, begin sorting at the lowest possible pressure with a wide bore nozzle and low flow rate. Collect a maximum of 25, 000 cells into each micro centrifuge tube so that the triol LS is not over diluted. Immediately after sorting vortex, each tube of cells captured in the triol ls.

In this first figure, a whole mount Euro genital tract at 15.5 days post coitus viewed ventrally under brightfield illumination is shown here. The same sample is viewed under fluorescence illumination To demonstrate the distribution of EGFP positive cells in the adrenal and medially located celiac ganglia as identified by their TH EGFP transgene expression. This lateral view of a 15.5 days post cotus TH EGFP sub dissected bladder shows fluorescence from transgene expression in the pelvic ganglia, the bladder wall, and the urethra.

In this dorsal view of the same EGFP positive cells are evident in the anterior dorsal urethra tissue dissociation to produce cell suspensions for flow sorting is a delicate balance between adequate enzymatic digestion and avoiding over digestion that can result in low cell variabilities. These photo micrographs show lower urogenital tract and intestinal tissues halfway through the dissociation time. As seen in these photos of appropriately digested tissue before and after manual, tri pieces of sub dissected organs are still clearly evident in tissues that are enzymatically treated for too long or at too high a concentration of enzyme.

However, the resulting suspension lacks any residual large pieces of tissue appropriate dissociation and manual filtering produces flow cytometry profiles that typically exhibit greater than 90%viable cells and those cells expressing the reporter in this viable population, cells retain high intensity EGFP expression. The black population is comprised of single cells that are dead and labeled by uptake of seven a a d fluorescence die. The gray population is comprised of singlet cells based on forward and side scatter that have excluded seven a a d, and are thus viable.

The green population is from the gated GFP positive area and is comprised of single viable cells that have excluded seven A a d, and that exhibit EGFP fluorescence. With this procedure, isolation of neuronal progenitors from other peripheral tissues like the sympathetic chain, dorsal root, ganglia or lung can be performed in order to answer questions about differences in gene expression profiles between populations or about the development of distinct lineages.