Immunofluorescent Staining of Live Cells for Nuclear Visualization
1. Dilute Hoechst 33342 solution to 5 - 10 μg/mL in complete medium immediately prior to use.
2. Add Hoechst 33342 solution to each sample and incubate at 37°C for 30 - 60 minutes. The stain time required is cell type dependent.
3. Remove Hoechst solution from cells at the end of the incubation period and add BD Pharmingen™ Stain Buffer (FBS) (Cat. No. 554656) or 1× PBS. Cells may also be analyzed without washing, but this may increase background from unbound dye.
4. Proceed to imaging.
Immunofluorescent Staining of Fixed Cells for Nuclear Visualization
1. Fix and permeabilize cells as desired.
2. Dilute Hoechst 33342 solution to 2 µg/ml in 1× PBS immediately prior to use.
3. Add 2ug/ml Hoechst 33342 solution to each sample at least 15 minutes before analysis.
4. Proceed to imaging.
Staining of Live Cells for DNA Content Analysis by Flow Cytometry
1. Obtain a single cell suspension.
2. Resuspend cells at 1x10^6 cells/mL or less in complete medium containing 5 - 10 μg/mL Hoechst 33342.
Note: Alternatively, Hoechst 33342 may be added directly to culture medium without pelleting if the culture cell density does not exceed 1x10^6 cells/mL.
3. Incubate at 37°C for 30 - 60 minutes.
a. The optimal cell density, concentration of Hoechst 33342, and stain time for DNA content analysis may vary by cell type. Assay conditions should be optimized in early experiments for best results.
4. Pellet cells by centrifugation and aspirate medium containing Hoechst 33342.
5. Resuspend cells in BD Pharmingen™ Stain Buffer (FBS) or 1× PBS and proceed to analysis by flow cytometry.
Staining of Fixed Cells for DNA Content Analysis by Flow Cytometry
1. Obtain a single cell suspension.
2. Treat cells on ice for 30 minutes with 70 - 80% ice-cold ethanol.
a. Ethanol fixation typically provides the most resolved histograms. However, this reagent has also been successfully used for DNA content analysis with the Transcription Factor Buffer Set (Cat. No. 562574) or BD Cytofix™ Fixation Buffer (Cat. No. 554655) and BD Phosflow™ Perm III (Cat. No. 558050) protocol.
3. Wash cells once with BD Pharmingen™ Stain Buffer (FBS).
4. Dilute Hoechst 33342 solution to 1 - 5 μg/mL in BD Pharmingen™ Stain Buffer (FBS) or 1× PBS immediately prior to use.
5. Stain cells for 15 minutes at a cell density of 1x10^6 cells/mL. No wash is necessary prior to analysis.
a. The optimal cell density and concentration of Hoechst 33342 for DNA content analysis may vary by cell type. Assay conditions should be optimized in early experiments for best results.
6. Proceed to analysis by flow cytometry.
This product is also available as a component of the Cell Cycle Kit (Cat. No. 558662). Please see the kit's Technical Data Sheet for a detailed protocol for the use of Hoechst Dye 333342 in conjunction with immunofluorescent staining of plated cells.
