A Comparative Study of Different Protocols for Isolation of Murine Neutrophils from Bone Marrow and Spleen
Neutrophils are considered as the main player in innate immunity. In the last few years, it has been shown that they are involved in different physiological conditions and diseases. However, progress in the field of neutrophil biology is relatively slow due to existing difficulties in neutrophil iso...
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| Format: | Article |
| Language: | English |
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MDPI AG
2023-12-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/24/24/17273 |
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| author | Khetam Sounbuli Ludmila A. Alekseeva Oleg V. Markov Nadezhda L. Mironova |
| author_facet | Khetam Sounbuli Ludmila A. Alekseeva Oleg V. Markov Nadezhda L. Mironova |
| author_sort | Khetam Sounbuli |
| collection | DOAJ |
| description | Neutrophils are considered as the main player in innate immunity. In the last few years, it has been shown that they are involved in different physiological conditions and diseases. However, progress in the field of neutrophil biology is relatively slow due to existing difficulties in neutrophil isolation and maintenance in culture. Here we compare four protocols based on density-gradient and immunomagnetic methods for isolation of murine neutrophils from bone marrow and spleen. Neutrophil isolation was performed using Ficoll 1.077/1.119 g/mL density gradient, Ficoll 1.083/1.090/1.110 g/mL density gradient and immunomagnetic method of negative and positive selection. The different protocols were compared with respect to sample purity, cell viability, yield, and cost. The functionality of isolated neutrophils was checked by NETosis analysis and neutrophil oxidative burst test. Obtained data revealed that given purity/yield/viability/cost ratio the protocol based on cell centrifugation on Ficoll 1.077/1.119 g/mL density gradient is recommended for isolation of neutrophils from bone marrow, whereas immunomagnetic method of positive selection using Dynabeads is recommended for isolation of splenic neutrophils. |
| first_indexed | 2024-03-08T20:41:46Z |
| format | Article |
| id | doaj.art-59f7d8bc76d542379e7b112442fbd935 |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-08T20:41:46Z |
| publishDate | 2023-12-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-59f7d8bc76d542379e7b112442fbd9352023-12-22T14:13:52ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672023-12-0124241727310.3390/ijms242417273A Comparative Study of Different Protocols for Isolation of Murine Neutrophils from Bone Marrow and SpleenKhetam Sounbuli0Ludmila A. Alekseeva1Oleg V. Markov2Nadezhda L. Mironova3Institute of Chemical Biology and Fundamental Medicine SB RAS, Lavrentiev Ave. 8, 630090 Novosibirsk, RussiaInstitute of Chemical Biology and Fundamental Medicine SB RAS, Lavrentiev Ave. 8, 630090 Novosibirsk, RussiaInstitute of Chemical Biology and Fundamental Medicine SB RAS, Lavrentiev Ave. 8, 630090 Novosibirsk, RussiaInstitute of Chemical Biology and Fundamental Medicine SB RAS, Lavrentiev Ave. 8, 630090 Novosibirsk, RussiaNeutrophils are considered as the main player in innate immunity. In the last few years, it has been shown that they are involved in different physiological conditions and diseases. However, progress in the field of neutrophil biology is relatively slow due to existing difficulties in neutrophil isolation and maintenance in culture. Here we compare four protocols based on density-gradient and immunomagnetic methods for isolation of murine neutrophils from bone marrow and spleen. Neutrophil isolation was performed using Ficoll 1.077/1.119 g/mL density gradient, Ficoll 1.083/1.090/1.110 g/mL density gradient and immunomagnetic method of negative and positive selection. The different protocols were compared with respect to sample purity, cell viability, yield, and cost. The functionality of isolated neutrophils was checked by NETosis analysis and neutrophil oxidative burst test. Obtained data revealed that given purity/yield/viability/cost ratio the protocol based on cell centrifugation on Ficoll 1.077/1.119 g/mL density gradient is recommended for isolation of neutrophils from bone marrow, whereas immunomagnetic method of positive selection using Dynabeads is recommended for isolation of splenic neutrophils.https://www.mdpi.com/1422-0067/24/24/17273neutrophil isolationFicoll density gradientimmunomagnetic separationpositive selectionnegative selection |
| spellingShingle | Khetam Sounbuli Ludmila A. Alekseeva Oleg V. Markov Nadezhda L. Mironova A Comparative Study of Different Protocols for Isolation of Murine Neutrophils from Bone Marrow and Spleen International Journal of Molecular Sciences neutrophil isolation Ficoll density gradient immunomagnetic separation positive selection negative selection |
| title | A Comparative Study of Different Protocols for Isolation of Murine Neutrophils from Bone Marrow and Spleen |
| title_full | A Comparative Study of Different Protocols for Isolation of Murine Neutrophils from Bone Marrow and Spleen |
| title_fullStr | A Comparative Study of Different Protocols for Isolation of Murine Neutrophils from Bone Marrow and Spleen |
| title_full_unstemmed | A Comparative Study of Different Protocols for Isolation of Murine Neutrophils from Bone Marrow and Spleen |
| title_short | A Comparative Study of Different Protocols for Isolation of Murine Neutrophils from Bone Marrow and Spleen |
| title_sort | comparative study of different protocols for isolation of murine neutrophils from bone marrow and spleen |
| topic | neutrophil isolation Ficoll density gradient immunomagnetic separation positive selection negative selection |
| url | https://www.mdpi.com/1422-0067/24/24/17273 |
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