Propidium Iodide (PI) or DAPI Staining of Unfixed Tissue Culture Cells for Flow Cytometry

Propidium Iodide (PI) or DAPI Staining of Unfixed Tissue Culture Cells for Flow Cytometry Linda Rodgers This protocol was adapted from “Flow Cytometry,” Chapter 16, in Cells (eds. Spector et al.). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 1998. This threevolume set is now out of print; however, some of the microscopy methods were republished in Basic Methods in Microscopy, by David L. Spector and Robert D. Goldman. INTRODUCTION This protocol describes a method for quantitative measurement of DNA in tissue culture cells using either propidium iodide (PI) or DAPI staining followed by flow cytometry. PI can be excited at 488 nm by the argon-ion laser, the most commonly used laser in flow cytometry. Alternatively, DAPI is best excited by a high-power UV laser, which is less commonly available. After staining, cells can be stored frozen for later sorting or analysis. MATERIALS Reagents Boiled RNase A, 10 mg/ml Citrate buffer DMSO (Optional, see Step 5) DNA staining/lysis solution (For PI staining; see Step 2.) NST-DAPI buffer (For DAPI staining; see Step 2.) Equipment Flow cytometer Nylon mesh, 35-μm pore size (Small Parts, Inc.) Page 1 of 3 Propidium Iodide (PI) or DAPI Staining of Unfixed Tissue Culture Cells for Flow Cytometry