TY - JOUR

T1 - A Comparative Study of Different Protocols for Isolation of Murine Neutrophils from Bone Marrow and Spleen

AU - Sounbuli, Khetam

AU - Alekseeva, Ludmila A

AU - Markov, Oleg V

AU - Mironova, Nadezhda L

N1 - This research was funded by the Russian Science Foundation (grant No. 22-14-00289) and by the Russian State funded budget project of ICBFM (grant number 121031300044-5).

PY - 2023/12

Y1 - 2023/12

N2 - 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.

AB - 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.

KW - Animals

KW - Mice

KW - Neutrophils

KW - Bone Marrow

KW - Spleen

KW - Ficoll

KW - Centrifugation, Density Gradient/methods

KW - Cell Separation/methods

UR - https://www.scopus.com/record/display.uri?eid=2-s2.0-85180707544&origin=inward&txGid=efa34f5b7c2f01b03e7838194c2af975

U2 - 10.3390/ijms242417273

DO - 10.3390/ijms242417273

M3 - Article

C2 - 38139101

VL - 24

JO - International Journal of Molecular Sciences

JF - International Journal of Molecular Sciences

SN - 1661-6596

IS - 24

M1 - 17273

ER -