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Updated: Aug 1, 2026
Preparation of Whole Bone Marrow for Mass Cytometry Analysis of Neutrophil-lineage Cells
Published on: June 19, 2019
DNA preparation from cryopreserved bone marrow cell samples
H Jørgensen1, P Hokland, E L Petersen
1Institute of Medical Microbiology, University of Aarhus, Denmark.
Abstract:
1. Quickly thaw cryopreserved MNCs (1 ml) in 9 ml PBS supplemented with DNase I immediately before use (final concentration: 0.1 mg/ml). 2. Incubate at 37 degrees C for 10 min. 3. Wash twice in PBS supplemented with 2% human AB serum at 4 degrees C. 4. Incubate with saturating amounts of selected MoAbs for 20 min at 4 degrees C. 5. Wash twice in PBS supplemented with 2% human AB serum at 4 degrees C. 6. Immunomagnetic bead separation of viable cells as described by Lea et al. (1). 7. DNA extraction. 8. Measure quality and yield of DNA by spectrophotometry. In conclusion, the DNA extraction method presented here, based on DNase I pretreatment of the cryopreserved cells followed by an immunological selection for viable cells, provides cell suspensions with close to 100% viability; thus, high-quality DNA can be extracted even from cryopreserved cell samples of low initial viability. Furthermore, using this method DNA analyses can be performed on selected cellular subsets if desired. We thus recommend this method for DNA analyses in hematological research when the only materials available are cryopreserved bone marrow samples of low viability.
Insights
This study presents a DNA extraction method for cryopreserved cells, enhancing viability using DNase I and immunomagnetic bead separation. This technique yields high-quality DNA from low-viability samples, crucial for hematological research.
Area of Science:
- Hematology
- Molecular Biology
- Cell Biology
Background:
- Cryopreserved samples often have low cell viability.
- Standard DNA extraction methods may be insufficient for compromised samples.
- High-quality DNA is essential for accurate molecular analyses in hematological research.
Purpose of the Study:
- To develop an optimized DNA extraction protocol for cryopreserved cells with low initial viability.
- To improve cell viability prior to DNA extraction.
- To enable reliable DNA analysis from challenging sample sources.
Main Methods:
- Cryopreserved cells were pretreated with DNase I to enhance viability.
- Immunomagnetic bead separation was employed to isolate viable cells.
- DNA was extracted and its quality/yield assessed by spectrophotometry.
Main Results:
- The method achieved near 100% cell viability post-treatment.
- High-quality DNA was successfully extracted from cryopreserved samples.
- The protocol allows for DNA analysis from selected cellular subsets.
Conclusions:
- The described DNA extraction method effectively recovers high-quality DNA from cryopreserved cells, even those with low viability.
- This technique is recommended for DNA analysis in hematological research using compromised bone marrow samples.
- The method facilitates targeted DNA analysis on specific cell populations.
