Lab 7: Cytokinesis

Myosin & tubulin expressing cells

2 MatTeks each per group

For ML-7, the final conc should be 75uM, so the 2X needs to be 150uM. For the Jasplak: the final conc should be 7uM so the 2X needs to be 14uM (but if 15 is easier, go with it). we use latrunculin at 5uM, and I think cytoD should be similar (I think I previously gave a range for cytoD of 1-10uM). Let's go with 5, so the 2X would be 10uM.

Inhibitors in non-CO2 medium (1 mL aliquots) each group does one (so make 5 of each):

• cytochalasin D Sigma C2618 or C8273

  • tubes have 40 µL 2.5 mM
  • label says add 460 µL to make 500 µL of 200 µM
  • need 10 µM (2x final working concentration)
  • 20 µL 2.5 mM
  • 5 mL non0CO2 medium
  • gives 5 1 mL aliquots at 10 µM

• Jasplakinolide – Santa Cruz biochemical, product sc202191 F

  • inducer of actin polymerization & stabilization
  • F-actin probe
  • MW = ~710
  • powder is stable frozen over a year
  • stock is stable 3-4 mo at -20C
  • Make 1 mM stock with 50 µg in 70 µL DMSO
  • Add 5 mL non-CO2 medium to make ~14 µM
  • 50 nM to 5 μM working range
  • minutes to hours incubation time

• ML-7 Sigma, 12764.

  • soluble 10mg/mL in 50% EtOH
  • MW = 452.74
  • 5 mg in bottle
  • add 1100 µL to make 10 mM solution
  • used at ~50 µM (1 µL/1mL) (Pat says 75 µM)
  • 75 µL 10 mM + 5 mL non-CO2 medium makes 150 µM
  • make 15 µL aliquots of 10 mM. Add 985 µL medium to make 150 µM