Cell assay, fix and stain (DAPI, Phalloidin)

This is the DRSC's basic protocol for fixation with paraformaldehyde 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.

Materials needed

  • 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

  • 43.25 mL PBS
  • 500 µl TritonX-100 (10%)
  • 6.35 mL PFA (32% paraformaldehyde)

PBT (.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

Protocol

  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.