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.

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:

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

best..