Isolation of Neural Stem Progenitor Cells from the Spinal Cord Periventricular Region of an Adult Rat
Trascrizione
To begin the isolation of rat neural stem cells, use micro-scissors to mince the dissected periventricular tissue into 1-cubic-millimeter pieces. Next, prepare the enzymatic dissociation buffer by first adding 5 milliliters of Earl's balanced salt solution to a vial of papain from a papain dissociation kit.
Place the vial at 37 degrees Celsius for 10 minutes, so that the papain is completely dissolved and the solution appears clear. Next, add 500 microliters of Earl's balanced salt solution to the vial of DNase from the dissociation kit, and gently mix.
Add 250 microliters of the DNase solution to the vial containing papain and gently mix. Next, add the minced tissue, approximately 0.2 grams, into the vial and place the vial on a rocker platform at 37 degrees Celsius for 45 minutes to one hour.
After incubation, triturate the mixture with a 10-milliliter pipette to dissociate any remaining tissue pieces to yield a cloudy cell suspension. Transfer the cell suspension into a sterile 15-milliliter conical tube, and centrifuge at 300 g's for 5 minutes at room temperature.
During centrifugation, prepare the ovomucoid solution by mixing 2.7 milliliters of Earl's balanced salt solution with 300 microliters of the reconstituted albumin ovomucoid inhibitor solution in a 15-milliliter conical tube. Add 150 microliters of the DNase solution previously prepared to the ovomucoid solution, and mix by inverting the tube.
Discard the supernatant from the pelleted cells and immediately resuspend the cell pellet in the diluted DNase albumin-inhibitor mixture. Next, prepare a discontinuous density gradient by adding 5 milliliters of albumin inhibitor solution to a 15-milliliter tube, and using a 5-milliliter pipette, gently and slowly layer the cell suspension on top of the albumin inhibitor solution.
Centrifuge the sample at 70 g's for six minutes at room temperature. Once finished, discard the supernatant, and resuspend the cell pellet in 1 milliliter of pre-warmed rat EFH medium that is prepared as described in the accompanying text protocol.
Count live cell density with a hemocytometer and plate the cells into a T25 culture flask at a density of 10 cells per microliter in EFH. Incubate the flasks at 37 degrees Celsius in 5% carbon dioxide and allow the cultures to grow undisturbed for one week to avoid aggregation of spheres.
