DNA Cell cycle analysis using Propidium Iodide

Developed by: Justin Meyers

Sample: Cell line or primary cells

Materials

  • Propidium iodide reagent (Sigma P-4170) 10mg make up 1mg/ml solution in DI water and store at 4C
  • Rnase A, (Sigma R-6513), 10mg, store at -20C

Procedure

Isolate cells

  1. Harvest cells, want good single cell suspension
  2. Count cells
  3. Dispense aliquots of 1 million cells into labeled tubes
  4. Add 5 ml PBS and centrifuge for 5 min at 400 x g.
  5. Re-suspend cell pellet in 300 ul PBS with gentle vortex

Fix cells:

  1. Add 700 ul ice cold ethanol (200 proof), slowly, while continuously vortexing.
  2. Incubate at 4C for 30 min to overnight
  3. Pellet cells at 2000 rpm for 5 min
  4. Re-suspend (vortex gently) cells in 250 ul of PBS
  5. Add 5ul of 10mg/ml Rnase A (final concentration being 0.2-0.5 mg/ml)
  6. Incubate at 37C for one hour
  7. Add 10ul of the 1mg/ml PI solution (final concentration is 10ug/ml)
  8. Keep in the dark for at least one hour until analysis
  9. Perform flow cytometry analysis on flow

Contact

Jill Hutchcroft
Flow Cytometry Facility Director
Phone: (765) 586-1130
Email: hutchcroft@purdue.edu

Purdue University, 610 Purdue Mall, West Lafayette, IN 47907, (765) 494-4600

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