The aim of this procedure is to isolate normal and cancer associated fibroblasts from fresh tissues. This is accomplished by first dissecting the mammary glands. The second step is to prepare mammary glands, single cell suspensions, next cell sustained with fibroblasts specific antibodies, as well as antibodies specific for other cell populations.
The final step is to perform fluorescence activated cell sourcing or facts. Ultimately, Q-R-T-P-C-R expression profiling of specific markers expressed in the sorted cell populations is used to assess the purity of the sorted cell populations. The main advantage of this technique over existing methods for fibroblast isolation is that it allows isolation of the majority of tissue fibroblasts with minimal contamination by immune cells or epithelial cells, while avoiding a tissue culture step that may change the gene expression profile of the fibroblasts.
The implication of this technique extend to all breast cancer therapy because understanding and identifying molecular targets may provide a novel Combinator approach to combat breast cancer progression, extend patient survival and ultimately help to improve and tailor individualized therapeutic options. In addition to your eye, Dr.Lena alone, the lab manager will be demonstrating the procedure To begin this procedure. Place the euthanized female mouse on its back on a styrofoam surface and use 25 gauge needles to firmly pin all four limbs.
Spray the mouse generously with 70%ethanol using forceps. Pull up the abdominal skin at the midline and make a small incision with sharp scissors starting from the incision. Cut the skin up to the neck of the animal while avoiding puncturing the abdominal thoracic cavities.
Cut the skin on the rear legs from the midline incision towards the leg, resulting in a Y shape. Next, pull skin away from the mouse body and pin down exposing the mammary glands attached to the underside of the skin. One of the most problematic thing in this procedure is to avoid taking muscle tissue.
In order to avoid this problem, I will take only the fourth and fifth mammary gland and not the second, which is in a close proximity to the muscle tissue. Use Q-tips to gently separate the memory gland from skin while cutting conjunctive tissue with small sharp scissors to separate the memory gland towards the spine of the mouse, remove any necrotic regions that are found. Place the dissected memory glands in PBS on ice.
The easiest way to obtain skin tissue without having to deal with hair removal is to use mouse ears. Use sharp scissors to cut off both ears from the euthanized mouse. Place the ears immediately in a digestion jar containing a small volume of PBS on ice.
To prepare a mammary gland single cell suspension used curved scissors to thoroughly mince the mammary glands in a digestion jar containing a small volume of PBS on ice, add 20 milliliters of collagenase solution to the mince tissue. This amount of collagenase solution will efficiently dissociate approximately five grams of memory or tumor tissue and ears from up to 15 mice. Place immediately on a stir plate in a 37 degree Celsius water bath and incubate for 15 minutes while stirring at a medium rate to stop the reaction at 30 milliliters of cold DMEM plus 10%FCS.
Next, strain the tissue and media mixture through a 70 micron cell strainer placed on top of a 50 milliliter conical tube. Centrifuge the conical tube for five minutes at 450 times G at four degrees Celsius. If the mice were not heart perfused with PBS before sacrifice, red blood cells in the sample will have to be lies before immune staining.
Cells must be blocked for endogenous FC prior to labeling fibroblasts and other cell populations. Resus suspend cells in one to three milliliter of fax buffer one, and then add FC block at a one to 50 dilution incubate on ice for 10 to 20 minutes. There is no need to wash off the FC block transfer 100 microliter cell aliquots to eend orph tubes to be used as an unstained control and single color controls.
Depending on the number of fluorescent antibodies used in this demonstration, two fluorescent antibodies will be used to label fibroblasts at anti PDGF receptor alpha. That is directly conjugated to a Fluor at a dilution of one to 50 to the sort sample as well as the single color controls to label other cell populations. Add the appropriate fluorescently conjugated antibodies to surface markers also at a one to 50 dilution.
Add antibodies also to appropriate single color control tubes, including antibodies for immune cells and epithelial cells is recommended in order to exclude any double positive populations and thus enhance the purity of isolated fibroblasts. Next, incubate the cells with antibodies for one hour on ice in the dark after an hour wash cells by filling tubes with fax buffer one and spinning for five minutes at 450 times G at four degrees Celsius, US aspirate sate and resus bend cells in fax buffer one to a concentration most appropriate for sorting with the fax sorter to be used. Transfer labeled cells into fax tubes with filter tops and filter cells into the tubes to prevent cell clumps, lumps to exclude dead cells.
A DPI to all samples except for the unstained control. Keep samples on ice during fax analysis. To begin this procedure, use the fact sorter software to prepare the fluorescent setting, acquisition setting and the hydrodynamic drop setting.
Vortex each sample briefly to resuspend cells before loading into the cell sorter. Start by analyzing the unstained sample in order to set the gating for the total cell population to gate out cell debris and to determine autofluorescence or background staining. Next, analyze the unstained sample plus DPI to determine and gate the population of live cells from the total cell population.
Analyze each single color control to determine and calibrate parameters such as compensation. Analyze the stain sample to define the position of gates for sorting. Once the parameters and gates for the desired cell populations have been set, begin sorting the labeled cell populations into eph tubes or 15 milliliter conical tubes containing culture medium or RNA lysis buffer.
Immediately after sorting is finished, spin cells down and transfer into fresh medium or RNA lysis buffer. Depending on the purpose of your experiment, if sorted, fibroblasts are to be cultured, always plate on collagen coated plates never plate directly on plastic, as this will result in activation of the fibroblasts leading to changes in their gene expression. Using PDGF receptor alpha as a marker for fibroblasts results in isolation, highly enriched populations of tissue fibroblasts.
In this example, single cell suspensions of mouse mammary glands were stained with PDGF receptor alpha and F four 80 antibodies. The fact sorting plot is shown in the left panel where P two is the fibroblasts gate, and P four is the macrophages gate. A post source analysis was performed to determine the purity of sorted fibroblasts shown in the right panel and macrophages shown in the middle panel.
This next figure shows an analysis of sourcing purity by Q-R-T-P-C-R of cell specific control genes for fibroblasts immune cells and epithelial cells. Results were normalized to two housekeeping genes, ga, DH and mgus, and relative expression to gap. DH is shown such analyses typically show 0.1 to 0.6%contamination.
This level of purity allows high quality transcriptome profiling of isolated fibroblasts. Quantification of PDGF receptor alpha expression in an unsorted population of mammary fibroblasts indicate that approximately 85%of total tissue fibroblasts in mammary glands are PDGF receptor alpha positive. I isolated.
Fibroblasts can be cultured directly after sorting, but may require a few days to recover. It is essential to perform all further experiments with low massage cells as primary fibroblasts undergo senescence during propagation. In culture, Once mastered, this technique can be done in four hours if it is performed properly.
If the thirded cells are designated to be cultured, it is imported to remember to perform all the procedure under sterile conditions. In this shoot, we did not perform a sterile sort. After watching this video, you should have a good understanding of how to isolate highly enriched population of fibroblasts from fresh tissue.
