|
▶Thaw 1 ml Matrigel on ice and keep cold = [20 wells (50 µl well)] ▶Put PBS0 = (Ca-Mg free PBS) on ice ▶Ad-DF+++ is: 500 ml Advanced DMEM/F12 (contain salbumin, and ITS ) 5 ml Glutamax 100 x 5 ml HEPES 1M 5 ml Pen/Strep (Bottle with Ad-DF +++ can be used, when stored cold, for 4 weeks) ▶PBS0/ EDTA: by adding 250 µl EDTA from 0,5 M stock to 50 ml PBSO = 2 mM and put on ice ▶PBS0/ FBS: by adding 5 ml FBS to 45 ml PBS0 for washing steps and put on ice Harvest mouse small intestine, put in 50 ml tube in PBS, if dirty flush once (not too much). In a 10 cm dish with PBSO remove fat from intestine and wash. Using small scissors cut the intestine open over the full length of the organ. Wash in PBSO. Open up the intestine a bit with tweezers and scrape of the villi using a haemacytometer coverslip leaving the crypts attached. Wash of the villi with PBSO and cut the intestine with scissors in small 2-4 mm pieces and transfer them to a 50 ml tube. Add 10 ml PBSO pipet up and down a few times with a 10 ml pipette remove the supernatant and add fresh PBSO. Repeat this 10-20 times until the supernatant is clear. (almost clear still bit cloudy after 20 washes) Add 25ml 2mM EDTA in PBSO and leave on ice for 30 minutes (or on rocking tube platform in cold room) While incubating with EDTA prepare or retrieve from freezer and fridge: to 10 ml Advanced DMEM/F12 +++ = + Hepes, Pen/step and Glutamax: After incubation with EDTA allow the small pieces to settle down and remove the supernatant. Add 10 ml PBSO 10% FBS and pipet up and down a few times (3-5) and collect the supernatant passing the 70 µM strainer, repeat this 3 more times using new strainers (these are the different crypt elution fractions) Spin down the crypt fractions at 800 rpm 5min The pellets were re-suspended with 10ml cold Ad-DF - GF and the tube is centrifuged with lower speed (600rpm, 2min), to remove single cells (mostly lymphocytes). Using a microscope check from each fraction the size of the crypts after going through the strainer, Estimate the number of crypt per fraction, you can pool the fractions (usually 2-3-4 the first one contains a lot of debris) first in a 50 ml tube and spin them at 600 rpm for 5 minutes at 4°C. Spin the desired amount of crypts again at 600 rpm for 5 minutes remove most of the supernatant and put the pellet on ice. (For 1 well in 24 wells plate we need 100-1000 crypts to be diluted in 50 µl Matrigel drop) For 20 wells add 1 ml thawed Matrigel to a pellet with 1000 – 10.000 crypts and pipet gently up and down using a cold 1000 µl tip (Matrigel should stay on ice like crypt pellet and tips). Add 50 µl drops without bubbles to the pre-warmed wells in 24 wells plates, wait 1-2 min at room temp and transfer to the incubator for 5-10 minutes. Add 500 µl of complete growth medium to the wells, as well as some PBS0 in the surrounding wells and transfer them back to the incubator (5%CO2). Typically, crypts start budding after 2-3 days in culture. Add fresh complete growth medium every 2-3 days. 500 µl for 2 days growth and 750 µl for 3 days growth. Passage outgrowing crypts after 7 days. Passaging crypts after 'sprouting' In 7-10 days the single crypts grow larger and start forming new crypts on the surface of the 'old' crypts. Crypts can grow 10x the size of the original isolated ones. Splitting ratio is ususally 1:5 - 1:6 Before passaging, narrow long Pasteur pipettes in the flame. Thaw Matrigel on ice. Prepare complete growth medium Pre-warm a 24 wells plate in the incubator (24 hours is best) Remove medium from Matrigel with crypts in 24 wells plate with p1000 pipett. Add 1ml cold Ad-DF+++ and break up the gel using a p1000 tip, transfer to 15 ml tube. Add another 1ml cold medium wash the well and transfer the medium to the 15 ml tube. Pre-wet the narrowed Pasteur pipet in Ad-DF and pipet up and down the slurry 10 x (with the pipet boy). keep cold during this procedure. After pipeting up and down a few times the slurry becomes less sticky and the crypts break up in little pieces but not too small (hardly visible). Add 2 ml cold Ad-DF and spin 5 min 600 rpm. Remove all the supernatant add 250 µl Matrigel pipet up and down and pipet 5 x 50 µl in 5 wells in pre-warmed 24 wells plate and transfer the plate to the incubator for 5-10 minutes. Add 500 µl complete growth medium and transfer the plate to the incubator. Re-feed every 2-3 days To avoid the crypts sticking inside the pasteurs pipette it’s possible to siliconize them.  |
|
|
