scientificprotocols authored about 3 years ago

Isolation of Lung Bud Endoderm

What you need:

  • E11-12 mouse embryos
  • DMEM with 5% fetal bovine serum
  • petri dishes for dissections and washes
  • Tyrode-Ringer's solution, pH 7.6-7.7 (recipe below)
  • pancreatin-trypsin solution (recipe below)
  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.

Solutions

Tyrode-Ringer's Solution, pH 7.6-7.7

component g/liter

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.

Collagen Gels

What you need:

  • Collagen, type I rat tail (cat no 40236, Collaborative Biomedical Products)
  • 10X DMEM, low glucose (cat no D6921, Sigma Cell Culture)
  • 0 .8M NaHCO3 in sterile water
  • four-well (1.5cm diameter/well) Nunc tissue culture dishes ( cat no 176740, Nalge Nunc International)
  • culture medium (for lung buds)
  • tissue culture incubator

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. Place at 37 ºC for approximately 10 minutes to allow complete polymerization.

  • Add culture medium to cover the bead of collagen and tissue and incubate the sample at 37º C.
  • Replace media daily.

DOI

Average rating 0 ratings