Can anyone share phalloidin staining protocol?

Malcolm Nobre

Hello Sreesada Parambath

Step 1. Prepare the A549 culture of adherent cells.

a. Grow A549 cells in a 96 well black wall/clear bottom plate until they reach confluence (70–80%).

b. You may also grow cells directly on coverslips inside a petri dish.

c. Aspirate cell culture medium (with care to avoid dislodging cells).

d. Wash once in PBS.

Step 2. Stain A549 cells with Alexa flour 594 phalloidin.

a. Fix cells in 3–4% formaldehyde in PBS at room temperature for 10–20 minutes.

b. Aspirate fixation solution and wash cells 2–3 times in PBS.

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

d. Add Alexa flour 594 phalloidin working solution. Incubate at room temperature for 20–90 minutes. Please note that the optimal concentration and the incubation time will vary depending on the particular cell type, fixation/sample prep conditions and/or permeability of the cells/ tissues to the probe.

e. Rinse cells 2–3 times with PBS, 5 min per wash.

f. Add mounting media to preserve fluorescence (and seal if you are using coverslips).

g. Observe the cells at Excitation/Emission: 581/609nm.

Some tips which you may follow during the experiment:

1) Avoid fixatives containing methanol or acetone as these disrupt the actin structure and prevent phalloidin staining.

2) Pre-incubating fixed cells with 1% BSA in PBS for 20 minutes may improve staining.

3) When staining coverslips, keep them in a covered container to minimize evaporation.

4) If cells do not appear healthy, you may add serum (2–10% range) to stain and wash the solutions.

Phalloidin will bind F-actin with high selectivity while Alexa Fluor 594 provides red fluorescence of unparalleled brightness and photostability. Demonstrating very little nonspecific staining, Alexa Fluor 594 phalloidin will allow high-contrast discrimination of actin staining.

Best.

Noor Rajeeb

Certainly! Here's a protocol for staining A549 lung cancer cells using Alexa Fluor 594 Phalloidin, which is commonly used to label F-actin in cells:

Materials:

A549 lung cancer cells
Cell culture medium
Phosphate-buffered saline (PBS)
Fixative solution (e.g., 4% paraformaldehyde in PBS)
Permeabilization solution (e.g., 0.1% Triton X-100 in PBS)
Blocking solution (e.g., 1% bovine serum albumin (BSA) in PBS)
Alexa Fluor 594 Phalloidin (concentration and dilution may vary depending on the manufacturer's instructions)
Mounting medium (e.g., mounting medium containing DAPI for nuclear counterstaining)
Microscope slides
Coverslips
Pipettes and tips
Centrifuge (if necessary)

Protocol:

Culture A549 lung cancer cells in appropriate cell culture medium until they reach the desired confluency or experimental condition.

Prepare the required solutions, such as fixative solution, permeabilization solution, and blocking solution, according to the concentrations mentioned above or as recommended by the manufacturer.

Harvest the A549 cells by washing the culture flask/dish with PBS and then detaching the cells using trypsin-EDTA or any other suitable cell detachment method. Collect the cells in a centrifuge tube and pellet them by centrifugation at an appropriate speed and duration (as per cell type and experimental needs).

Remove the supernatant carefully, and resuspend the cell pellet in the fixative solution. Incubate the cells in the fixative for about 10-15 minutes at room temperature to immobilize and preserve the cellular structure.

Wash the fixed cells with PBS to remove the fixative solution.

Permeabilize the cells by adding the permeabilization solution and incubating for about 5-10 minutes at room temperature. Permeabilization helps in the entry of the staining reagents into the cells.

Wash the cells again with PBS to remove the permeabilization solution.

Prepare the staining solution by diluting the Alexa Fluor 594 Phalloidin according to the manufacturer's instructions. Typically, a recommended dilution is around 1:200-1:500, but this can vary depending on the specific product.

Remove the excess PBS and apply the staining solution to the cells, ensuring all cells are covered. Incubate the cells with the staining solution for an appropriate period, usually 30 minutes to 1 hour, at room temperature or as suggested by the manufacturer.

After staining, carefully wash the cells with PBS to remove the unbound staining solution.

If desired, you can counterstain the nuclei by incubating the cells with a suitable nuclear stain, such as DAPI, following the manufacturer's instructions. This step is optional but helps visualize the cell nuclei along with the actin cytoskeleton.

Finally, mount the stained cells onto microscope slides using a suitable mounting medium. Place a drop of mounting medium on a clean microscope slide, gently transfer the stained cells onto the drop, and carefully cover them with a coverslip, avoiding air bubbles.

Allow the mounting medium to dry, and then seal the coverslip edges with clear nail polish or an appropriate mounting sealant.

The stained cells are now ready for visualization under a fluorescence microscope. Use appropriate filter sets to visualize Alexa Fluor 594 and DAPI (if used) fluorescence.

Remember to always follow the specific instructions provided by the manufacturer of the Alexa Fluor 594 Phalloidin and any other reagents used in.

best..

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