The overall goal of this procedure is to isolate, enrich, and maintain mouse medulloblastoma cells with stem cell properties. This is accomplished by first dissecting the medulloblastoma tissue from a genetically modified mouse. The second step of the procedure is a mechanical and enzymatic dissociation of the dissected tumor tissue.
The third step of the procedure is centrifugation of the dissociated tumor cells, followed by removal of the enzymatic solution. The final step of the procedure is plating the tumor cells in gelatinized tissue culture dishes. Ultimately, results can be obtained that show formation of highly proliferative clon genic, and multipotent medulloblastoma stem cells that can undergo multiple passages and propagate secondary tumors by allografting assays.
The main advantage of this technique over the existing method, such as neuros sphere culture method for cancer stem cell is that this technique allows equivalent access to nutrients, therefore reduce spontaneous differentiation. In apoptosis, My sparing modelo blasts stoma are often rented hydrocephalic and display typical neurological symptoms including posterior paralysis and a failure to regain posture. When overturned.
Use stainless steel four inch surgical scissors to remove as much hair and muscle tissue as possible from the decapitated head of a euthanized mouse. With medulloblastoma, clean the skull surface with a wipe soaked in 95%Ethanol under a dissecting microscope, use a sterile razor blade to cut an opening along the midline of the skull with fine tweezers. Remove skull tissue exposing the entire brain.
The cerebellar of healthy adults display well-defined hemispheres and vermes with cerebellar of tumor bearing. Mice are often enlarged. An amorphous with a smooth surface and conspicuous blood vessels.
Use fine tweezers to remove the tumor. Place the tumor in a sterile 60 millimeter dish of ice cold one times PBS without magnesium or calcium for one to two minutes. Transfer the tissue to a four well plate containing approximately four times the tissue volume of 50%Accutane in one times PBS use fine scissors to fine mince the tissue into small pieces of less than one millimeter diameter.
Tritrate with a one milliliter pipe peppermint for three minutes. Incubate at 37 degrees Celsius for four minutes. Then tritrate with a one milliliter pipe tum for another three minutes.
Yielding a suspension of single cells and small cellular aggregates. Dilute the suspension threefold with one times PBS in a 50 milliliter Falcon tube. Pellet the cells at 1000 times gravity for five minutes.
Resuspend the pellets in three milliliters of fresh neural stem cell culture. Medium plate all of the cells from one pellet onto a gelatinized 60 millimeter paria dish for enhanced attachment. Incubate at 37 degrees Celsius for one week for the initial plating feed and maintain cultures as detailed in the accompanying text.
One week after initial plating, there will be many colonies on the plate to enrich the tumor cell population. Replace the medium with fresh neural stem cell culture.Medium. Use a one milliliter pipe peppermint to mechanically detach colonies from the dish and tri rate for four minutes.
To yield a cell suspension. Check under a light microscope to ensure that large cell clumps have been dissociated. Dispense all of the cell suspension onto a plain gelatinized 60 millimeter dish Culture at 37 degrees Celsius for one week.
Changing the medium every other day. Monoculture without unattached tumor cells and blood cells is generally reached after five serial rep platings. Each tumor cell line established from an animal can be kept for more than 20 passages.
To prepare slides for immunofluorescence staining seed, approximately one milliliter containing two to four times 10 to the five cells over gelatinized glass cover slips in each well of a six well plate add differentiation. Medium if desired to the cells on the same day as plating. Proceed to immunofluorescent staining as described in their accompanying text.
This series of brightfield images demonstrate a typical progression of changes over a 10 day period of culturing medulloblastoma cells from a cell suspension into clonally expanded colonies. This brightfield image shows the typical appearance of established medulloblastoma cells. After several passages, the cells remain mostly bipolar with a high nuclear to cytoplasmic ratio.
Acetylated tubulin staining is in red. Most of the tumor cells are cycling as shown by strong KI 67 expression in green smoothened M two YFP marks cells expressing constitutively, active smoothened and hence sonic hedgehog pathway activity. All of the established medulloblastoma cell lines express YFP and most of them coex express high levels of various neural stem cell markers such as NEST in and SOX two YFP positive tumor cells switched from E-G-F-B-F containing serum free medium to DMEM 10%FBS significantly altered their morphology and differentiated into various cell types, including TJ one positive or new N positive neurons.
GFP positive ASTROGLIA cells or CPA's positive oligodendrocytes Once mastered this technique can be done within 30 minutes if performed properly.
