Isolation of Lung Bud Endoderm
What you need:
Tyrode-Ringer's Solution, pH 7.6-7.7
NaCl 8.0 KCl 0.3 NaH2PO4.5H2O 0.093 KH2PO4 0.025 NaHCO3 1.0 Glucose 2.0 Pancreatin/Trypsin Solution
component g/20 ml final concentration
pancreatin 0.50 2.5% trypsin 0.10 0.5% polyvinylpyrrolidone(PVP) 0.10 0.5% Tyrode-Ringer's Solution 20 ml 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.
Place collagen, 10X DMEM and on ice. Also chill the four-well dishes in the rfridge before use.
Mix 45 ul collagen and 5 ul 10X DMEM. Mix well on ice. The collagen will not gel if kept cold.
Add 1.9 ul of NaHCO3. Mix well. This step begins polymerization of the collagen gel.
Place at 37 ºC for approximately 10 minutes to allow complete polymerization.