Cell assay, fix and stain (DAPI, Phalloidin)

This is the DRSC's basic protocol for fixation with formaldehyde and staining with DAPI and/or phalloidin. It is also appropriate for fixation prior to immunostaining, although some antibodies, cell types, target proteins, etc. might require a modified protocol.

Note: Formaldehyde and paraformaldehyde are toxic and carcinogenic chemicals and should be carefully handled and disposed of. Please make sure proper hazardous waste disposal protocols are in place before using these chemicals in experiments.

16% formaldehyde protocol

(updated March 2020)


  • 1x PBS
  • 0.1% PBT: 1x PBS + 100% Triton X-100
  • 16% formaldehyde
  • Fixing solution: dilute 16% formaldehyde to 4% formaldehyde using PBS
  • Staining solution: 0.1% PBT + 1:1000 Phalloidin + 1:2000 DAPI (or stains of choice at correct concentrations)


1. Remove media from the 384-well plate using a multichannel aspirator connected to a vacuum. If you are concerned that cells may be lifted and removed by the aspirator, remove media by inverting the plate onto paper towels and patting dry. There will still be a little media left in each well.

2. Add 1x fixing solution. This can be done two ways:

  • Make a 2x fixing solution (8% formaldehyde in PBS). If the amount of media left in each well after vacuuming is known, add 2x fixing solution at equal volume, which will result in a 1x fixing solution.
  • Add a large volume of 1x fixing solution (~90uL), then remove. Add another large volume. This will ensure the small amount of media is removed with the first wash and all the solution left in the wells are at 1x.

3. Incubate the plate for 20 minutes at room temperature on an orbital shaker at low speed.

4. Remove the fixing solution using the aspirator or inverting the plate onto paper towels, then add 75uL PBT. Incubate the plate for 5 minutes at room temperature on the orbital shaker.

  • Remove the PBT, then repeat this step two more times for a total of three washes.

5. Remove the last wash of PBT, then add 90uL of the staining solution. Repeat.

6. Incubate the plate overnight on a shaker at 4 degrees.

7. Remove the stain, then add 90uL of PBT to each well.

  • Repeat this step twice more for a total of three washes.

8. Remove the PBT, then add 90uL of PBS to rinse. Repeat for a total of two rinses.

9. Add 50uL of PBS and image. Store at 4 degrees.

32% paraformaldehyde method


  • 100% TritonX-100
  • 1x PBS
  • 32% paraformaldehyde
  • DAPI (1000x)
  • Alexa Fluor 488 phalloidin

Solutions needed

  • 10% TritonX-100 (prepare the night before): 45 mL phosphate-buffered saline (PBS) + 5 mL 10% TritonX-100
  • Fixation solution (see below)
  • DAPI and phalloidin in PBS (1:1000 dilution)

Fixation solution

  • 3.25 mL PBS
  • 500 µL TritonX-100 (10%)
  • 6.35 mL PFA (32% paraformaldehyde)

PBT (0.1%)

  • 50 mL PBS
  • 500 µL TritonX-100 (10%)

For one 384-well plate, we use 12.5 µl DAPI (1000x) and 12.5 µl phalloidin


  1. Prepare .1% PBT, fresh fix solution (4% PFA in .1% PBT), and staining solution (DAPI and phalloidin in PBS at a 1:1000 dilution).
  2. Remove media from cells by inverting plates (e.g. over paper towels) and gently shaking ("shake-out method").
  3. Dispense 25 µl/well of 1x PBS, such as with a multi-channel pipet, Matrix WellMate, or other liquid handling device (hereafter we will refer to the WellMate), into each 384-well plate.
  4. Remove PBS by shake out method.
  5. Dispense 25 µl/well of fix solution with WellMate and fix cells for 25 minutes.
  6. Remove fix by shake out method.
  7. Dispense 20 µl/ well of PBT with WellMate. Allow to sit for 5 minutes, shake out and repeat two more times for a total of 3 washes.
  8. Remove PBT by shake out method.
  9. Dispense 25 µl/well staining solution with WellMate. Allow to incubate for 25-30 minutes.
  10. Remove staining solution by shake out method.
  11. Dispense 20 µl/ well of 1x PBS. Allow to sit for 5 minutes, shake out and repeat two more times for a total of 3 washes.
  12. Remove PBS by shake out method.
  13. Add 50 µl/well of 1x PBS to keep the cells hydrated while imaging.