10 slides w/ DAPI

(1 per microscope + 2 extra)

Micrometers

5 or 6 slides each:

• Rhodamine Phalloidin "1G"
• Stained for rat anti-tubulin + goat anti-rat FITC (or GFP-tubulin cells) "2B"
• Acridine orange stained for nucleoli "3G"
• Stained with with mouse anti-Golgi + goat anti-mouse 568 "4G" (formaldehyde fixed)
• Stained with rabbit anti-ZO1 + goat anti-rabbit Cy (cell-junction) "5G"

20 fixed coverslips for phalloidin staining

3 MatTeks per group for mitotracker and ?

aliquots of

• phalloidin
• mitotracker
• non-CO2 medium

Finish looking at slides, discuss cell division

10 slides DAPI + tubulin

2 MatTeks per group each:

• LLCPK with GFP tubulin
• LLCPK parentals

NUC-Blue for staining chromosomes in live cells (Invitrogen R37605 or Fisher NC0291762), 1 bottle per station

• Remove from culture dish after ~10 minutes. Replace stain medium with fresh medium. Otherwise it gets brighter and brighter as movie goes on

Non-CO2 medium

4 MatTeks per group of LL-GFP-tubulin

Double-strength inhibitors, made up in non-CO2 medium:

• STLC Sigma 164739 (Fisher 50-703-1833), MW = 363.47

• need 1 mL aliquots 10 µM in non-CO2 medium for students

• Make 100 mM stock:

  • 1 g in bottle.
  • Mix with 27.5 mL DMSO
  • heat to 65C (~1 hr), vortex
  • if necessary, filter sterilize to remove insoluble particles.
  • save as 1 mL aliquots. (dilute 1:10,000 to use) (dilute 1:100 to make 1 mM)

• Make 1 mM stock from the 100 mM stock

  • 10 µL 1mM stock
  • 990 µL DMSO
  • vortex
  • it will crystallize in the refrigerator. Warm and vortex to redissolve it.

• Make 17 mL 10 µM in medium for students

  • 170 µL 1 mM stock
  • medium to 17 mL
  • aliquot

• Make 10 µL aliquots of 1 mM stock

  • label says to add 990 µL medium to make 10 µM working solution

from Alyssa:madke STLC in DMSO and that she heated to 42C, and vortexed. She says it went from clear to yellow/brown. we use the reagent at micromolar - 1-10 µM. Her stock solution was 1mM.

students will be adding 1 mL of inhibitor to 1 mL medium in dish; the ready-to-use 1 mL (in medium) should be at least 2µM

• Taxol (20 µM final)
• soluble in DMSO, not water.
  • students pick either Taxol or nocodazole, so make only 10 each
  • each tube has 10 µL 10 mM (if it precipitates, add 10 uL DMSO and vortex, then add 480 uL medium or buffer)
  • add 490 µL to make 200µM in two tubes!
  • mix with medium in 15 mL conical to make 10 mL at 20 µM

• Nocodazole (3.3 µM final)

  • each tube has 3 µL 33mM
  • add 97 µL to tube to make 100 µL of 1 mM
  • 33 µL 1mM + 10 mL medium = 3.3 µM

• Nuc Blue from Invitrogen (or Fisher NC0291762), 1 bottle per station

  • Remove from culture dish after ~10 minutes. Replace stain medium with fresh medium. Otherwise it gets brighter and brighter as movie goes on

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

Students limited to 3 MatTeks of one of the following:

• GFP tubulin
• tubulin-myosin cells

Plus their choice of inhibitors:

• latrunculin
• taxol
• nocodazole
• jasplakinolide
• ML-7
• STLC

Report on mitosis experiments