3.3: Direct fluor

3.3: Direct fluor kdorfman Tue, 12/06/2011 - 15:57

Direct Fluorescent Labeling of Organelles

CHECK THIS FOR HBS vs PBS!!

Cells

3t3, LLCPk

Fixed in paraglut for phalloidin
- 8 3t3 (can be fixed as late as the morning of lab)
- 8 LLCPk (can be from pre-semester prep)
Live on small diameter MatTek dishes for Mitotracker and ceramide
- 8 3t3
- 8 LLCPk

Reagents

Serum-free non-CO2 medium

6 mL/group

2 mL WARM for Mitotracker in live cells in MatTek dishes

3 mL COLD for ceramide

PBS (or HBS?)

~3 mL to rinse coverslips treated with Mitotracker

~3 mL to rinse MatTek dishes

Phalloidin

50 µL/ coverslip (15 min at RT)

2 coverslips per group

100 µL/group

aliquoted to make 100 µL. Directions on tube.

Mitotracker

diluted in serum-free non-CO2 medium

1 mL/group

100 nm working concentration

stock is 1 mM in DMSO

Mix 1 µL in 10 mL

Aliquot ~1.2 mL/group

Either view live, or fix in 3.2% formaldehyde (in PBS) NO DETERGENT

Ceramide

Invitrogen N22651

fluorescent marker for Golgi

Follow instructions attached: Add 150 µL sterile H2O to the 5 mg in the bottle. (Makes 0.5mM (=500 µM) in BSA)

Aliquot 10 µL, and freeze

Working concentration is 5 µM

Add 990 µL HBS to 10 µL in tube

OR 99µL 10x HBS + 891 µL water

Aliquot ~120 µL per group (makes 8 aliquots)