User:Mar/Notebook/2007-7-26

= Migration of Suspended GE Cells (1) =

Proof-of-Concept experiment to show whether suspended cells from GE can migrate at all, part. toward cortical explants.

suspension

 * 1) GE explants sit in DPBS-Mg/Ca for 30' in the incubator in a Petri dish (to gently break down cell-to-cell binding)
 * 2) DPBS replaced with DPBS/medium (can be repeated)
 * 3) trituration in 1.5mL of DPBS (with syringe using gradually thinner needles: 26, 22, 18)
 * 4) spinning 2000xg for 2', removing supernatant, resuspending in 1ml of NeuroBasal (+N+B+G) medium (deviation: DPBS used)
 * 5) counting: 26.8 mln/mL, diluted to 25mln/mL {#cells in all 16 fields x 10e4 x dilution}
 * 6) suspending in MatriGel (1+2 with medium, i.e. 140µL MG + total 70µL of suspension (cells + adjustment with medium):
 * 7) * #1 70µl  + 0µL medium     + 140µL MatriGel (=25mln/mL * .070mL /.210mL = 8.3mln/mL)
 * 8) * #2 35µl  + 35µL medium    + 140µL MatriGel
 * 9) * #3 17.5µl + 52.5µL medium + 140µL MatriGel
 * 10) * #4 8.75µl + 61.25µL medium + 140µL MatriGel
 * 11) 200µL of suspension spread over coverslip
 * 12) cortical explants, having been incubated in medium on a dish, were placed onto the suspensions with glass Pasteur pipette.
 * 13) Explants covered with a drop of pure MatriGel
 * 14) after 15 min incubation, cultures covered with medium 2mL/dish at 6:00pm
 * 15) each dish imaged at 10x in brightfield at 7:00pm

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