from Hogan Lab
Isolation of Lung Bud Endoderm
What you need:
1. Dissect E11-12 lungs in DMEM and 5% serum on ice. Young lungs (E11 to 11.5) are best.
2. Wash the lungs 3 x 5 minutes on ice in Tyrode-Ringer's Solution, pH 7.6-7.7 to optimize physiological conditions for pancreatin-trypsin activity.
3. Incubate whole lungs for appoximately 5 minutes in pancreatin-trypsin solution on ice. The time required will vary depending on the batch of PT.
4. Wash lungs 3 x 5 minutes in DMEM + serum to saturate pancreatin-trypsin activity.
5. Using tungsten needles, dissect off the mesenchyme and cut off the buds. Alternatively, cut off the buds first and remove the mesenchyme surrounding them.
6. Place into matrigel matrix and culture for three to five days (see accompanying protocol). Isolated endoderm will not survive without growth factor added to the culture medium.
Tyrode-Ringer's Solution, pH 7.6-7.7
component g/20 ml final concentration
Suspend by mixing for about 30 minutes. Centrifuge at 3000Xg in the Sorvall hanging bucket rotor for 5 minutes. Filter sterilize the supernatant using .8 micron Millipore filter(s). Store in aliquots at -80 C. This process is a big pain and time consuming, mainly because the supernatant does not filter efficiently and the Millipore filters must be switched often.
What you need:
Tissues to be cultured are placed into 50 ul drops of collagen in four-well Nunc dishes. For each treatment, make a separate 50 ul aliquot of collagen gel as described in the protocol below and store on ice. Directly before adding samples, add the NaHCO3 and mix well. It is necessary to prepare each collagen gel separately because addition of NaHCO3 makes the collagen polymerize quickly.
1. Place collagen, 10X DMEM and on ice. Also chill the four-well dishes in the rfridge before use.
2. Mix 45 ul collagen and 5 ul 10X DMEM. Mix well on ice. The collagen will not gel if kept cold.
3. Add 1.9 ul of NaHCO3. Mix well. This step begins polymerization of the collagen gel.
4. Add the 50 ul collagen mixture to a chilled four-well dish.
5. First prepare a bed of collagen and allow it to set at 37ºC. After it has polymerized, add your sample to the bed of collagen and add cold unpolymerized collagen in a layer over the top of the sample.
6. Place at 37 ºC for approximately 10 minutes to allow complete polymerization.