The overall goal of this procedure is to culture myogenic precursor cells from teleosts to undergo myogenesis in vitro. This is accomplished by first dissecting the epaxal muscle from the organism. The second step is to mechanically dissociate the isolated tissue.
Next, the tissue is enzymatically dissociated to disperse the desired cells. The final step is to plate the isolated myogenic precursor cells on Lamin in substrate. Ultimately in vitro.
Myogenesis is used to show the effects of various treatments on changes in gene regulation and expression, protein expression, and cell phenotype changes. This video will demonstrate how to set up a primary culture system from teleost Myogenic precursor cells. Visual demonstration of this technique is imperative as some of the dissociation steps may be difficult to learn.
Proper and adequate dissociation is vital for isolating an adequate cell pellet for later culture. To begin, prepare all of the media and solutions needed for the procedure. Next, assemble additional items needed for tissue isolation.
Autoclaving beforehand where necessary. Be sure to prepare additional materials for each person assisting with the dissection process. Other items to have on hand include 70%ethanol, a balance sterile, 50 milliliter conical tubes and ice for each five grams of tissue to be dissected.
Aliquot 25 milliliters of isolation medium into a sterile 50 milliliter conical tube. After weighing and recording the mass number, the tubes and place them on ice. When ready, retrieve a fish to be used in tissue dissection and submerge in 70%Ethanol for 30 seconds before placing it on water repellent autoclave paper directly behind the erla uses scalpel to make a superficial shallow incision, grasp the skin at the incision and pull towards the tail to remove the scales and skin.
Next, excise the axel fast glycolytic white muscle of the fish from both sides. Avoiding the slow oxidative red muscle located near the lateral line. Place the muscle into isolation, medium and discard the remainder of the fish.
Continue to collect tissue until a sufficient quantity of muscle is obtained during dissection, ensure that pooled muscle tissue remains on ice to maintain cell viability. To begin pour one tube of muscle tissue into a glass Petri dish. Use two scalpels in a back and forth motion to mince the tissue until the tissue slurry can be easily removed.
With a 25 milliliter pipette, place the tissue slurry back into its original tube. Repeat these steps until all tissue has been mechanically dissociated. When all of the tissue is processed centrifuge for five minutes afterwards, discard the supernatant and wash the tissue with fresh media before another round of centrifugation.
Once washed thoroughly resus, suspend the tissue and incubated with collagenase solution For 60 minutes at 18 degrees Celsius with gentle rocking. At the end of the incubation centrifuge the tubes to pellet the cells. After washing twice, resus, suspend the tissue in wash medium.
Next, triturate the tissue homogenate until it passes in and out of a 10 milliliter pipette with relative ease. Repeat this process with a five milliliter pipette and then again with a 16 gauge metal cannula attached to a syringe. Once thoroughly homogenized, pellet the tissue and decant the supernatant.
Re suspend the tissue in trips in dissociation medium and incubate for 20 minutes at 18 degrees Celsius. Afterwards, place the tubes in the centrifuge for a brief spin following centrifugation. Decant the supernatant into four equal volumes of isolation.
Media to be stored at four degrees Celsius to the remaining pellet. Repeat the trips and dissociation steps just demonstrated and combine the resulting supernatant. With that collected previously.
Dispense the final mixture into 50 milliliter, conical tubes and centrifuge. After centrifugation, remove the supernatant taking care not to disturb the cell. Pellet pipette two milliliters of complete medium into each tube.
To dissolve, combine the resuspended cells into one 50 milliliter conical tube. Rinse each tube with one milliliter of complete medium. Adding the rinse to the pool of resuspended cells.
Tritrate the tissue with a metal cannula and syringe five to 10 times. After filtering the cell suspension, add a sufficient amount of complete medium to equal 50 milliliters and centrifuge. Retrieve the cells, remove the supernatant and add five milliliters of complete medium.
Once Resuspended, remove a small sample to determine the number of viable cells. Using a hemo cytometer. Remove laminin solution from poly L lysine treated plates.
After diluting the cells to the needed concentration, plate them onto the prepared plates and seal. Place the sealed plates into a chilling incubator set at the appropriate temperature. 24 hours Post seeding myogenic precursor cells or MPCs should be visibly attached to the laminin substratum, while originally appearing more compact.
MPCs from zebrafish adopt similar morphologies to rainbow trout. Over four days of culture, myo tubes should form within six to nine days of culture. In culture, MPCs and myoblasts are shown to express myo D one.
As myoblasts differentiate into myo tubes, they will begin to express myo genin. These data show cell proliferation during culture using BRDU in incorporation. After watching this video, you should have a good understanding of how to isolate and culture.
Myogenic precursor cells from a variety of fish species After its initial development in cell Monets. This technique paved the way for researchers and muscle biology to explore new avenues in muscle biology and other organisms and other telio like danios, as well as other amphibians or amphibians like Axel.Lots.
