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
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