Labeling Nuclear DNA with Hoechst 33342

Adapted from Imaging: A Laboratory Manual (ed. Yuste). CSHL Press, Cold Spring Harbor, NY, USA, 2010.

INTRODUCTION

A number of fluorescent stains are available that label DNA and allow easy visualization of the nucleus in interphase cells and chromosomes in mitotic cells. One advantage of Hoechst 33342 is that it is membrane permeant and, thus, can stain live cells. Hoechst 33342 binds to adenine-thymine-rich regions of DNA in the minor groove. On binding to DNA, the fluorescence greatly increases. This protocol describes the use of Hoechst 33342 to label nuclear DNA of cells grown in culture.

RELATED INFORMATION

Hoechst 33342 can also be used to stain fixed cells by substituting Hoechst 33342 for DAPI in the protocol described in Labeling Nuclear DNA Using DAPI (Chazotte 2011a). Autofluorescence from endogenous cellular molecules such as the reduced forms of nicotinamide adenine dinucleotide or flavin adenine dinucleotide can interfere with imaging by reducing the signal-to-noise ratio. This occurs when the excitation and/or emission wavelengths of the probe and the autofluorescing molecules are similar, e.g., frequently with excitation wavelengths 4] for 20 min), and by comparing the experimental images with unlabeled control slides. Avoid fixation with glutaraldehyde, because it can increase interference from cellular autofluorescence, most frequently at wavelengths

This protocol assumes that the cells of interest were grown on glass microscope coverslips immersed in small Petri dishes containing culture medium. Generally, labeling conditions vary by cell type, and it might be necessary to alter the protocol for a particular use. To mount cells labeled using the technique described here, see Mounting Live Cells onto Microscope Slides (Chazotte 2011b).

MATERIALS

Reagents

Cells of interest, grown on coverslips

Hoechst No. 33342 (10 mg/mL in H₂O stock solution; Invitrogen H1399)

Store stock solution at 4°C, protected from light.

Phosphate-buffered saline (PBS)

Prepare PBS with added CaCl₂ and MgCl₂ (PBS + ). This solution allows cells to adhere to each other and to the substrate. If cells are in medium containing no Ca 2+ or Mg 2+ , they will round up and detach from the substrate.

Equipment

Cell culture dishes, sterile

Microscope, fluorescence, equipped with an ultraviolet (UV) filter set

For confocal microscopy laser excitation, use a UV laser or, if sufficiently intense, the UV line of an argon-ion laser.

METHOD

Do not allow the cells to dry out at any time during the protocol.

• 1. Dilute the Hoechst stock solution 1:100 in H₂O for use in labeling.
• 2. Aspirate the cell medium from cells grown on coverslips. Rinse the cells three times with PBS + .
• 3. Incubate the cells in the Hoechst labeling solution (from Step 1) for 10-30 min at room temperature.
• 4. Aspirate the labeling solution. Rinse the cells three times in PBS + .
• 5. Mount the coverslips as described in Mounting Live Cells onto Microscope Slides (Chazotte 2011b).
• 6. Image the cells (λ_ex ~353 nm, λ_em ~483 nm for Hoechst 33342).

ACKNOWLEDGMENTS

I thank my wife, Nancy, and my daughter, Bryanna, for their patience while I was writing this article. I dedicate this article in memory of my mother, Cozette Chazotte, 1919-2009.