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):
 * 8) * #1 165µl  + 33µL medium    + 402µL MatriGel (=25mln/mL * .070mL /.210mL = 8.3mln/mL)
 * 9) * #2 81µl  + 117µL medium    + 402µL MatriGel
 * 10) 100µL of suspension spread over coverslip
 * 11) cortical explants, having been incubated in medium on a dish, were placed onto the suspensions with glass Pasteur pipette.
 * 12) 100µL of suspension spread on top of cortical explants as a second layer
 * 13) after 15 min incubation, cultures covered with medium 2mL/dish
 * 14) 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