Table of Contents:

Endoderm Prep (to strip off and replate the outer layer of endoderm cells from mature embryoid bodies)

Endoderm Prep

1.  Sterilize thick walled plastic 9 ml centrifuge tubes by filling with EtOH.  Use about one tube for each 150 mm dish of embryoid bodies (EBs) or equivalent.

2.  Remove 7 or 8 day embryoid bodies (with visible endoderm) from petri dishes and put in 50 ml conical to settle.  Wash plates with PBS and add this to tubes.

3.  Allow EBs to settle and wash twice with PBS

4.  Add 5 ml trypsin/EDTA (GIBCO) to EBs and transfer them to a 100 mm petri dish.  Leave at 37 deg C. for 15-20 minutes to desintegrate.

5.  Add 5 ml of complete medium.  Roughly (but not so rough as to lyse cells) pipette the EBs up and down.  Your goal is to obtain a single cell suspension.  You can monitor this by checking them in the inverted microscope.

6.  Spin down the cells, and bring up in 4 ml complete medium for each tube you will be using.

7.  Pour out EtOH from tubes and gently flame tops (if you flame too much, tubes will melt and will not fit into the adaptors for spinning.)

8.  In sterile plastic 9 ml tubes add 3 mls of lymphocyte separation medium (LSM, from Organon Teknika in Durham, NC), 1 ml of a 1:1 mixture of LSM and medium, and 4 mls of your cell suspension.  Do this carefully so the layers remain separate!

9.  Spin at setting #5 on a clinical centriguge for 10-12 minutes.

10.  Remove tubes.  You should see an upper band of endoderm cells in the 50/50 interlayer.  This often appears more like a diffuse cloud than a tight band.  The stem cells and EB fragments will band lower, just above the LSM layer.  Remove the top band of endoderm cells with a sterile pasteur pipette, spin down and resuspend in complete medium.

11.  Count cells in a hemocytometer.  Endoderm cells will look bigger and rougher than the small, smooth stem cells, so you can estimate the purity of your prep.  Yields of endoderm cells vary enormously among different cell lines.  Plate as needed.  We put 1 x 10⁶ vcells in a 60 mm dish, 5 x 10⁵ in a 35 mm dish.  We put 1 x 10⁶ cells in a 35 mm dish with 50 ul Cytodex beads (Pharmacia).