5.2 2014

Cells

  • 3t3 on coverslips - 2 per group
  • 3t3 on MatTek dishes (small diameter)
  • LLCPk GFP-alpha tubulin on coverslips 1 per group
  • LL parentals on coverslips 2 per group

Reagents

HBS-BSA

TMR-dextran to stain endosomes in cells on coverslips
Invitrogen D3308, 10 mg
Make 10 mg/mL stock (add 1 mL to the bottle)

TMR-dextran Working concentration = 0.05 mg/mL in medium

  • 2 coverslips at 50 µL/coverslip (=~110 µL/group) + 2 live cells at 100 µL/mattek
  • Aliquot 5.2 µL 10 mg/mL
  • Label says to add 98.8 µL to make 0.5mg/mL working soloution

Lysotracker (Invitrogen L-7528 - 20 x 50 µL)

  • 50 - 75 nM
  • stock = 1 mM in DMSO
  • aliquot 10 µL in 2 mL tubes, so they can make 2 mL medium
  • 30 min - 2 hours incubation warm.

Transferrin in Fe-HBS-BSA

  • Alexa-Fluor 488 transferrin (Invitrogen T13342, MW = ~80KD) stock is 5 mg/ml (=62.5 µM). Final concentration = 1 µL.
  • Each group does 2 50-µL treatments (need extra for pipetting error)
  • Make 1.2 mL: 19.2 µL AF-488-Tf + 1.1808 mL Fe-HBS-BSA
  • Aliquot 165 µL

    if new transferrin stock is needed add 1 mL sterile distilled water to the new bottle.