實驗概要
The method we use is based on work of Dr. Hazel Cheng, at the University of Toronto and works for both colon and the small intestine.
First we excise the whole colon from cecum to rectum. This is easily done after openning the abdomen by grasping the cecum with forceps and pulling until it is straight. Then cut close to the cecum and the rectum.
主要試劑
0.075 M KCl solution containing 20 mM EDTA
Pancreatin
Agar
RPMI 1640 Medium
Sodium Hypochlorite
主要設備
Knives
Petri dishes, 9 cm Flasks, 25 cm2 Scalpels, holder #4, blade #22 Syringe, 10 ml Needles, 21 gauge Movette pipette
實驗步驟
1. First we excise the whole colon from cecum to rectum. This is easily done after openning the abdomen by grasping the cecum with forceps and pulling until it is straight. Then cut close to the cecum and the rectum.
2. Flush the colon using a 10 ml syringe and a large needle, preferably with the point cut off, eg. #16. This is to remove the contents, which you can see flush out. The flushing solution probably does not matter. We use either saline or the incubation solution which is used in the 4th step.
3. Invert the colon. This is easy once you have done it a few times, but tricky at first. There are several different techniques that people use in the lab. One is to pass a crochet hook or disecting needle with a hooked end through the colon, and hook on to the outside at the other end. Then just draw the needle back thus turning the colon inside out. A second way is to scrunch the colon onto the end of a pair of fine forcepts until they protrude from the other end, grab the end with the tip of the forceps (usually with the help of another pair), and then , again, draw the forceps back, turning the colon inside out. Actually, I expect one could simply slit the colon after flushing but because of the work we do with the small intestine, we are practiced at the inversion. Possibly the 5th step would be more difficult on a slit colon.
4. Place the inverted colon in a 0.075 M KCl solution containing 20 mM EDTA (the incubation solution). Leave for 10 - 20 minutes. When the next step works well, the timing is right, so you can test every 5 minutes or so. See the next step for criteria.
5. Crack the colon. This was developed by David Blakey in my lab many years ago. Take a 1 ml tuberculin syringe without the needle. Put the end of the syringe at the end of the colon and pull the plunger sharply. The colon should resist briefly and then enter the syringe with a snap - "crack". If the colon will not enter, leave it longer in the incubation solution. All but very old mouse colons will work in our experience. If the colon enters without a snap, try grabbing it in the middle instead of the end. If it still enters without a snap, reduce the inubation time on the next mouse. Force the colon in and out of the syringe as fast as possible about 5 times. You can see when it is flaccid and the solution is not getting more turbid. Sometimes one or two cracks are enough. More than 10 are a waste of time.
6. Isolate the DNA. You can pellet the material first if you like. If you examine the suspension under the microscope you will see a mixture of crypts, which resemble eppindorf tubes, broken crypts, and single cells. If you look after the first crack, you will see mostly intact crypts, which is fun. There are probably, almost certainly, some lymphocytes from the Peyer's patches and some myofibroblasts from the supporting substructure contaminating the preparation, but we are certain these are less than 5% and normally less than 1% of the cells.
