Homogenize cells or tissues in buffer, followed by sonication. An amount of the cell suspension is mixed with Hoechst 33258 and the fluorescence is determined. Source: Animal Cell Culture: A Guide to Basic Techniques 5th Edition

Operation method

Scheme 21.3 DNA assay with Hoechst 33258

Principle

Homogenize cells or tissues in buffer, followed by sonication. An amount of the cell suspension is mixed with Hoechst 33258 and the fluorescence is measured.

Move

makings

Non-sterile:

Buffer: 0.05 mol/L NaPO4; 2.0 mol/L NaCl, pH 7.4 , containing 2 X ^l0-3 mol/L EDTA .
Hoechst33258; in buffer, use lug/ml for DNA above 100ng/ml,0.lug/ml for 10~100ng/ml.

Operating Procedure

1. Homogenize cells in buffer with a Potter homogenizer at 1X ¹⁰⁵ cells/ml for 1min.

2. Ultrasonicate for 30s.

3. Dilute cells at 1 : 10 with Hoechst 33258 and buffer.

4. Detect fluorescent radiation at 492nm with excitation at 356nm, using calf thymus DNA as the standard.

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