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