Phalloidin Conjugates

1. Prepare samples in microplate wells
2. Remove liquid from samples in the plate
3. Add Phalloidin-Fluorescein Conjugate solution (100 μL/well)
4. Stain the cells at room temperature for 20 to 90 minutes
5. Wash the cells
6. Examine the specimen under microscope with FITC filter

Warm the vial to room temperature and centrifuge briefly before opening.

The solution should be stable for at least 6 months if store at -20 °C. Protect the fluorescent conjugates from light, and avoid freeze/thaw cycles.

Note: Phalloidin is toxic, although the amount of toxin present in a vial could be lethal only to a mosquito (LD50 of phalloidin = 2 mg/kg), it should be handled with care.

Add 1 µL of Phalloidin-Fluorescein Conjugate solution to 1 mL of PBS with 1% BSA.

Note: The stock solution of phalloidin conjugate should be aliquoted and stored at -20 °C. protected from light.

Note: Different cell types might be stained differently. The concentration of phalloidin conjugate working solution should be prepared accordingly.

1. Perform formaldehyde fixation. Incubate cells with 3.0–4.0 % formaldehyde in PBS at room temperature for 10–30 minutes.

   Note: Avoid any methanol containing fixatives since methanol can disrupt actin during the fixation process. The preferred fixative is methanol-free formaldehyde.

2. Rinse the fixed cells 2–3 times in PBS.

3. Optional: Add 0.1% Triton X-100 in PBS into fixed cells for 3 to 5 minutes to increase permeability. Rinse the cells 2–3 times in PBS.

4. Add 100 μL/well (96-well plate) of Phalloidin-Fluorescein Conjugate working solution into the fixed cells, and stain the cells at room temperature for 20 to 90 minutes.
5. Rinse cells gently with PBS 2 to 3 times to remove excess phalloidin conjugate before plating, sealing and imaging under microscope with FITC filter set.