(Skip to menu.)

Staining Protocol for Mitochondria and Actin Filament

A. Preparation

1. Splitting Adherent Cells

Purpose

Reduce cell density and replate cells.

Materials

• Cell Medium (see Cell Culture protocol)
• PBS Ca-Mg free (Irvine Scientific #9240 - $12.04/500ml)
• Trypsin EDTA 1X (Irvine Scientific #9340 - $6.32/100ml)
• Chambered Coverglass (Nalge Nunc International #155380 - $78.01/pack of 16)

Protocol

1. Aspirate away all media.
2. Add 10 ml of PBS CA-Mg free and aspirate.
3. Add 1ml of trypsin.
4. Put in a 37ºC CO2 incubator for 3-5 minutes. Cells should lift in 5-10 minutes maximum. Check with a microscope.
5. Add 5 ml of medium and gently pipette up and down.
   
   
   If you just want to split the cell in a tissue culture dish, for later use:
6. Depending on the initial cell density, put 1ml to 6ml of the medium + trypsin solution in a tissue culture dish (10 cm in diameter) and add some medium to have a final volume of 10ml.
   
   
   Or if you want to replate the cells in a chambered cover glass for staining:
7. Aspirate medium and trypsin leaving ~ 200 µl behind.
8. Add 10 ml of medium and resuspend the cells.
9. Dilute the cell suspension 1:10 to use within 1-3 days and 1:40 to use within a week.
10. Put 2 ml of the diluted solution into each side of the chambered cover glass.

2. Preparation of the dyes

MitoTracker Green:

The final concentration of the dye in the 2ml-chamber should be 100nM. To do so, prepare some 5 µl-vial with:

• 0.2 µl 1mM MitoTracker Green stock solution (in anhydrous DMSO)
• 4.8 µl anhydrous DMSO

One vial will be used for each 2ml-chamber.

Phalloidin Red (rhodamine):

Use 2 units per 2ml-chamber. The stock solution is a 200 units/ml solution. Prepare tubes with 10 µl of this solution. One tube will be used for each 2ml-chamber.

B. Staining cells

1. Vitally label the mitochondria in living cells

1. Pipette a little bit of the medium in the tube containing the 5 µl MitoTracker Green dye and mix.
2. Put everything into the chambered cover glass and mix gently.
3. Put in the 37ºC CO2 incubator for 15-45 minutes.
4. Aspirate away the labeling solution and rinse with fresh prewarmed medium. Repeat this step 2 times.
   
   The coverglass is then ready for imaging.

2. Visualize F-actin distribution in fixed, adherent cells

1. Aspirate the medium and fix cells for 30 minutes in fresh 4% PFA (~ 2 ml) (leave the cover glass under fume hood and protect them from the light with aluminum foil).
2. Aspirate the PFA and rinse 2 times in PBS for 5 minutes each.
3. We will keep one chamber with non-labeled cell. In every chamber but one, add the phalloidin (to the PBS) and wait 30 minutes.
4. Rinse 2 times in PBS for 5 minutes each.
5. Post-fix in 4% PFA for 15 minutes.
6. Rinse 3 times in PBS for 5 minutes each.
   
   The coverglass is then ready for imaging.

Note 1: Step 1 and 3 can be done together. So step 1 would be fix + stain then step 4 to 6 as described before. The quality of the staining is better in the first case, but doing step 1 and 3 together is a good solution to save time.

Note 2: The mitochondria can be visualized in fixed, adherent cells too. The protocol is the same as for the F-actin filament, but using MitoTracker Green instead of Phalloidin Red.

back

Navigation