Hello, my name's Annaa from the laboratory of Professor Ika Egar in the Department of Pediatric Oncology at the University Children's Hospital, Esen, Germany. Today we will show you a procedure for preparing extracts of 11 days old prenatal embryonic chicks. Put DMEM on ice to cool it down to at least four degrees Celsius.
We use DMEM glutamate from GIB co prepared sterile tubes by filling them half. With the MEM, wash the eggs with 70%E ethanol use X that have been incubated for 11 days at 100 degree Fahrenheit in humidified incubator. Be sure to let the ethanol be absorbed for at least 30 seconds.
After applying it carefully dry the eggs with soft paper towels. Clean a plastic or glass plate with ethanol. Open the eggs.
Carefully put the embryo into the plate. Do not destroy the EC or any little vessels. Use sharp clean precision pin sets to dissect out the embryos.
First, look for the umbilical cord.Again. Do not destroy the yac or any small vessels while searching for the umbilical cord. Once you found the umbilical cord, follow to its beginning and dissect it.
After opening the clear wrapping above the embryo, carefully take the Embryo out of the EC without destroying it. Place the embryos in sterile, minimal essential medium on ice. We use DMEM glutamate from gyp Co.Take the plunger out of a sterile 60 milliliter siren.
Put approximately 10 embryos at a time Into the siren. Put the plunger into the siren. Maturate the embryos by pushing down the plunger.
Be careful not to use too much pressure otherwise you will lose maturated material. Collect the maturated embryos in the prepared tubes at the ratio of one-to-one embryos to the MEM 10 embryos. Should produce approximately 25 milliliters of volume.
Shake the mix of the ratio of one-to-one embryos to EMEM for 45 minutes at four degrees Celsius. Carefully transfer the mix into the centrifugation tubes. Add the rile hyaluronidase days.
We use the R Hay days from Boin testis from sigma. Tear the centrifuge tubes with a special AC accuracy weighing machine. It is very important to take care of the three points after decimal point.
They have to be equal. Otherwise, you risk having problems with the centrifuge because of the high speed needed to centrif ate the mix If necessary, adjust inequalities by pipetting the missing amount of the MEM. The Rotor should have been inside a fridge or a cooling chamber overnight to cool it down to four degrees Celsius.
The centrifugation tubes must be closed very tightly. Otherwise you risk having problems to get them out of the rotor. After the centrifugation, Fill the rotor with the tubes and close it very tightly in advance.
Cool down the centrifuge to four degrees Celsius. Use vacuum In every Case. Wait until the rotor has caught its final speed.
If anything strange happens, immediately stop the centrifuge. Press the brake button to stop the centrifugation. This is after a TI I 45 rotor has run for six hours at a speed of 45, 000 rounds per minute.
Carefully take out the tubes. Do not shake them at all for this could cause an increasement of the upper dingy liquid layer inside the tubes. Notice The three phases.
At the top there is dinghy liquid with fatty pieces. In the middle, there is clear liquid, and at the bottom you can see the pellet. Be very careful to catch the clear phase out of the middle of the tube.
Do not touch the pellet at all as this would lead to contamination of the extract and also to obstruction of the filter system. First, use a 0.45 micrometer filter connected to a vacuum pump In the second Step, filter the attained liquid through a zero point 22 micrometer filter. Also connected to vacuum pump Qu the extract inster Tubes.
Finally store the tubes at minus 80 degrees Celsius.
