A Comparative Study of Different Protocols for Isolation of Murine Neutrophils from Bone Marrow and Spleen
Abstract
:1. Introduction
2. Results
2.1. Protocol Selection for Neutrophil Isolation
2.2. Characterization of the Samples Isolated from Bone Marrow and Spleen
2.3. Comparison of Purity, Viability, and Yield of Neutrophil Samples Isolated from Bone Marrow and Spleen Using Different Protocols
2.4. Ability of BM-Derived Neutrophils Isolated by the 2FLG Protocol and Splenic Neutrophils Isolated Using the IPS Protocol to Produce NETs
2.5. ROS Production in BM-Derived Neutrophils Isolated Using the 2FLG Protocol and Splenic Neutrophils Isolated Using Dynabeads in Response to Stimulation
3. Discussion
4. Materials and Methods
4.1. Mice
4.2. Bone Marrow Cell (BMC) Isolation
4.3. Splenocyte Suspension Preparation
4.4. Preparation of Ficoll Solutions of Different Densities
4.5. Neutrophil Isolation
4.5.1. Ficoll 1.077/1.119 g/mL Density Gradient (Two Layer Density Gradient Protocol, 2FLG Protocol)
4.5.2. Ficoll 1.083/1.090/1.110 g/mL Density Gradient (Three Layer Density Gradient Protocol, 3FLG Protocol)
4.5.3. Immunomagnetic Methods
Immunomagnetic Negative Selection (INS Protocol)
Immunomagnetic Positive Selection (IPS Protocol)
4.6. Neutrophil Characterization
4.6.1. Sample Purity
4.6.2. Cell Viability
4.7. Neutrophil Extracellular Trap Visualization
4.8. Neutrophil Oxidative Burst Test
4.9. Statistical Analysis
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Method of Isolation | Sample Purity, % | Viability, % | General Yield, ×106 | Neutrophil Yield, ×106 | Cost |
---|---|---|---|---|---|
2FLG | 72.4 ± 1.7 | 89.8 ± 1.4 | 8.6 ± 2.8 | 6.3 ± 2.1 | the lowest |
3FLG | 76.7 ± 5.0 | 91.0 ± 2.9 | 0.5 ± 0.1 | 0.4 ± 0.04 | middle |
INS | 80.3 ± 0.3 | 94.3 ± 0.8 | 10.7 ± 1.1 | 8.6 ± 0.9 | the highest |
IPS | 99.3 ± 0.3 | 91.6 ± 0.3 | 4.3 ± 1.4 | 4.3 ± 1.4 | high |
Method of Isolation | Sample Purity, % | Viability, % | General Yield, ×106 | Neutrophil Yield, ×106 | Cost |
---|---|---|---|---|---|
2FLG | 8.6 ± 1.2 | 74.0 ± 7.0 | 18.1 ± 1.9 | 1.5 ± 0.3 | the lowest |
3FLG | 10.5 ± 1.7 | 86.0 ± 4.0 | 0.8 ± 0.1 | 0.08 ± 0.02 | middle |
INS | 48.6 ± 4.3 | 89.6 ± 2.0 | 6.5 ± 2.4 | 3.3 ± 1.5 | the highest |
IPS | 98.7 ± 0.5 | 88.5 ± 4.5 | 0.7 ± 0.25 | 0.69 ± 0.25 | high |
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Sounbuli, K.; Alekseeva, L.A.; Markov, O.V.; Mironova, N.L. A Comparative Study of Different Protocols for Isolation of Murine Neutrophils from Bone Marrow and Spleen. Int. J. Mol. Sci. 2023, 24, 17273. https://doi.org/10.3390/ijms242417273
Sounbuli K, Alekseeva LA, Markov OV, Mironova NL. A Comparative Study of Different Protocols for Isolation of Murine Neutrophils from Bone Marrow and Spleen. International Journal of Molecular Sciences. 2023; 24(24):17273. https://doi.org/10.3390/ijms242417273
Chicago/Turabian StyleSounbuli, Khetam, Ludmila A. Alekseeva, Oleg V. Markov, and Nadezhda L. Mironova. 2023. "A Comparative Study of Different Protocols for Isolation of Murine Neutrophils from Bone Marrow and Spleen" International Journal of Molecular Sciences 24, no. 24: 17273. https://doi.org/10.3390/ijms242417273