User:Mar/Notebook/2007-8-3

Migration of Suspended GE Cells (2)

Next Proof-of-Concept experiment to show whether suspended cells from GE can migrate at all, part. toward cortical explants. Modification: Oregon Green to label the cortical explants

suspension

1. dissected GE explants wait under NBasal in an incubtor
2. GE explants sit in DPBS-Mg/Ca for 15'-30' in the incubator in a Petri dish (to gently break down cell-to-cell binding)
3. DPBS replaced with DPBS/medium (can be repeated)
4. trituration in 1.5mL of DPBS (with syringe using gradually thinner needles: 26, 22, 18)
5. spinning 2000xg for 2', removing supernatant, resuspending in 1ml of NeuroBasal (+N+B+G) medium
6. counting: 30.9 mln/mL {#cells in all 16 fields x 10e4 x dilution}
7. suspending in MatriGel (1+2 with medium, i.e. 140µL MG + total 70µL of suspension (cells + adjustment with medium):
   • #1 165µl + 33µL medium + 402µL MatriGel (=25mln/mL * .070mL /.210mL = 8.3mln/mL)
   • #2 81µl + 117µL medium + 402µL MatriGel
8. 100µL of suspension spread over coverslip
9. cortical explants, having been incubated in medium on a dish, were placed onto the suspensions with glass Pasteur pipette.
10. 100µL of suspension spread on top of cortical explants as a second layer
11. after 15 min incubation, cultures covered with medium 2mL/dish
12. each dish imaged at 10x in brightfield at 6:00pm ()

day 0

imaging of baseline

day 1 (Fri)

• suspended cells not migratory
• activity of cortical explants inversely proportional to the density of suspension:
  • explants "silent" in the highest density
  • explants send migrating cells and/or fibers gradually more as density decreases

day 4 (Mon)

• some suspended cells show migratory morphology
• activity of cortical explants inversely proportional to the density of suspension, although all are active

day 6 (Wed)

• fixation in 4% PFA at 10am