5.2 2015

Cells

• 2 coverslips 3t3 per pair for TMR-dextran
• 2 LL-GFP-tubulin in MatTek dishes per pair for lysotracker & endosome movement
• 3t3 in MatTek for live studies

Reagents

• HBS-BSA (1 mg/mL). per pair ~7mL:

  • 6 mL for 3 rinses live cells
  • 1 mL for nocodazole
  • 0.1 mL for Transferrin

• Nocodazole 1 µM in HBS-BSA 1 mL per pair

  • aliquots are 3 µL 3mM in DMSO
  • add 97 µL to make 1mM stock
  • dilute 1:1000 in HBS-BSA (10 µL in 10 mL)

• Transferrin in Fe-HBS-BSA to label live cells 100 µL/pair

  • Stock is 5 mg/ml (=62.5 µM)
  • Working conc = 1 µM
  • 0.016 µL Tf/µL solution
  • 1 mL = 16 µL Tf + 984 µL Fe-HBS-BSA

• Lysotracker in non-CO2 medium 1mL/pair

  • stock is 1 mM
  • working solution is 50 - 75 nM
  • Dilute 7.5:100,000 = 0.75 µL/10 mL

• TMR-dextran (Invitrogen D3308, 10 mg) in non-CO2 medium

  • 100 µL to stain in dish
  • 100 µL to stain 2 coverslips
  • 10 mg/mL stock, in 8.8 ML aliquots
  • 0.05 mg/mL working concentration
  • aliquot ~210 µL per pair

• CO2 medium (to rinse and return to incubator)

• Non-CO2 medium (for imaging)

• PBS for rinsing

• 3.7% paraformaldehyde in PBS for fixation: 3 mL/pair