Limits...
Rapid immunomagnetic negative enrichment of neutrophil granulocytes from murine bone marrow for functional studies in vitro and in vivo.

Hasenberg M, Köhler A, Bonifatius S, Borucki K, Riek-Burchardt M, Achilles J, Männ L, Baumgart K, Schraven B, Gunzer M - PLoS ONE (2011)

Bottom Line: Their lack or dysfunction is associated with severe health problems and thus the analysis of PMN physiology is a central issue.Most importantly, cells obtained by this method are non-activated and remain fully functional in vitro or after adoptive transfer into recipient animals.This method should thus greatly facilitate the study of primary murine PMN in vitro and in vivo.

View Article: PubMed Central - PubMed

Affiliation: Institute for Molecular and Clinical Immunology, Otto-von-Guericke University Magdeburg, Magdeburg, Germany.

ABSTRACT
Polymorphonuclear neutrophils (PMN) mediate early immunity to infection but can also cause host damage if their effector functions are not controlled. Their lack or dysfunction is associated with severe health problems and thus the analysis of PMN physiology is a central issue. One prerequisite for PMN analysis is the availability of purified cells from primary organs. While human PMN are easily isolated from peripheral blood, this approach is less suitable for mice due to limited availability of blood. Instead, bone marrow (BM) is an easily available reservoir of murine PMN, but methods to obtain pure cells from BM are limited. We have developed a novel protocol allowing the isolation of highly pure untouched PMN from murine BM by negative immunomagnetic isolation using a complex antibody cocktail. The protocol is simple and fast (~1 h), has a high yield (5-10*10⁶ PMN per animal) and provides a purity of cells equivalent to positive selection (>80%). Most importantly, cells obtained by this method are non-activated and remain fully functional in vitro or after adoptive transfer into recipient animals. This method should thus greatly facilitate the study of primary murine PMN in vitro and in vivo.

Show MeSH

Related in: MedlinePlus

Cell composition of murine BM.The bone marrow of 8–10 weeks old female C57BL/6 mice was isolated and after erythrocyte lysis the percentage of the particular surface marker positive cells on total living cells was assessed. Gray bars indicate the antibodies which were used in the neutrophil-isolation cocktail presented in this study. Percentages are means of at least three independent experiments.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3044161&req=5

pone-0017314-g001: Cell composition of murine BM.The bone marrow of 8–10 weeks old female C57BL/6 mice was isolated and after erythrocyte lysis the percentage of the particular surface marker positive cells on total living cells was assessed. Gray bars indicate the antibodies which were used in the neutrophil-isolation cocktail presented in this study. Percentages are means of at least three independent experiments.

Mentions: To establish a protocol that would allow the negative isolation of murine neutrophils from bone marrow we first had to identify good candidate surface markers which are present on marrow resident cells but absent from neutrophils. It was essential, that all markers were expressed on the surface of cells since we wanted to isolate living cells without the need for fixation and permeabilization. To this end we used a selection of fluorescently labeled monoclonal antibodies binding to a wide spectrum of different surface markers and measured the amount of antibody binding cells in normal bone marrow by flow cytometry. (Fig. 1) shows the relative percentage of cells and the identities of the labeled antigens for which we could detect binding of specific antibodies in normal murine bone marrow. The next step required to identify those antigens that were absent on neutrophils and, at the same time, non-overlapping with each other in terms of cellular specificity. Thus, e.g. CD14 was not a good marker, as it was 100% overlapping with F4/80, while F4/80 stained also other cells that were not detected by CD14. A cocktail containing anti-CD14 antibodies would thus not yield as pure neutrophils as one containing F4/80. Specifically with F4/80 we also found, that the antibody clone C1:A3-1 did not yield good results, while clone BM8 was very effective. Finally, all potential candidates were checked in double stainings against the neutrophil-specific marker Gr-1 stained by the antibody RB6-8C5 [13], [14] and were only taken, if Gr-1 positive cells were not stained by a defined antibody. With these selection criteria we finally identified a cocktail consisting of antibodies specific for 6 different antigens, namely CD5 (T cells), CD45R/B220 (B cells), CD49b/DX5 (NK cells), CD117 (Mast cells and hematopoietic stem cells), F4/80 (clone BM8, macrophages) and Ter119 (erythrocytes). A mixture of antibodies against these 6 types of cells/antigens was then labeled with biotin. Bone marrow harvested by flushing of femoral bones was freed of erythrocytes by short-term hypotonic lysis as described [15] and then incubated with the biotinylated antibody mixture. The optimized amount of each antibody in the cocktail used for the number of bone marrow cells in a sample is given in table 1.


Rapid immunomagnetic negative enrichment of neutrophil granulocytes from murine bone marrow for functional studies in vitro and in vivo.

Hasenberg M, Köhler A, Bonifatius S, Borucki K, Riek-Burchardt M, Achilles J, Männ L, Baumgart K, Schraven B, Gunzer M - PLoS ONE (2011)

Cell composition of murine BM.The bone marrow of 8–10 weeks old female C57BL/6 mice was isolated and after erythrocyte lysis the percentage of the particular surface marker positive cells on total living cells was assessed. Gray bars indicate the antibodies which were used in the neutrophil-isolation cocktail presented in this study. Percentages are means of at least three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3044161&req=5

pone-0017314-g001: Cell composition of murine BM.The bone marrow of 8–10 weeks old female C57BL/6 mice was isolated and after erythrocyte lysis the percentage of the particular surface marker positive cells on total living cells was assessed. Gray bars indicate the antibodies which were used in the neutrophil-isolation cocktail presented in this study. Percentages are means of at least three independent experiments.
Mentions: To establish a protocol that would allow the negative isolation of murine neutrophils from bone marrow we first had to identify good candidate surface markers which are present on marrow resident cells but absent from neutrophils. It was essential, that all markers were expressed on the surface of cells since we wanted to isolate living cells without the need for fixation and permeabilization. To this end we used a selection of fluorescently labeled monoclonal antibodies binding to a wide spectrum of different surface markers and measured the amount of antibody binding cells in normal bone marrow by flow cytometry. (Fig. 1) shows the relative percentage of cells and the identities of the labeled antigens for which we could detect binding of specific antibodies in normal murine bone marrow. The next step required to identify those antigens that were absent on neutrophils and, at the same time, non-overlapping with each other in terms of cellular specificity. Thus, e.g. CD14 was not a good marker, as it was 100% overlapping with F4/80, while F4/80 stained also other cells that were not detected by CD14. A cocktail containing anti-CD14 antibodies would thus not yield as pure neutrophils as one containing F4/80. Specifically with F4/80 we also found, that the antibody clone C1:A3-1 did not yield good results, while clone BM8 was very effective. Finally, all potential candidates were checked in double stainings against the neutrophil-specific marker Gr-1 stained by the antibody RB6-8C5 [13], [14] and were only taken, if Gr-1 positive cells were not stained by a defined antibody. With these selection criteria we finally identified a cocktail consisting of antibodies specific for 6 different antigens, namely CD5 (T cells), CD45R/B220 (B cells), CD49b/DX5 (NK cells), CD117 (Mast cells and hematopoietic stem cells), F4/80 (clone BM8, macrophages) and Ter119 (erythrocytes). A mixture of antibodies against these 6 types of cells/antigens was then labeled with biotin. Bone marrow harvested by flushing of femoral bones was freed of erythrocytes by short-term hypotonic lysis as described [15] and then incubated with the biotinylated antibody mixture. The optimized amount of each antibody in the cocktail used for the number of bone marrow cells in a sample is given in table 1.

Bottom Line: Their lack or dysfunction is associated with severe health problems and thus the analysis of PMN physiology is a central issue.Most importantly, cells obtained by this method are non-activated and remain fully functional in vitro or after adoptive transfer into recipient animals.This method should thus greatly facilitate the study of primary murine PMN in vitro and in vivo.

View Article: PubMed Central - PubMed

Affiliation: Institute for Molecular and Clinical Immunology, Otto-von-Guericke University Magdeburg, Magdeburg, Germany.

ABSTRACT
Polymorphonuclear neutrophils (PMN) mediate early immunity to infection but can also cause host damage if their effector functions are not controlled. Their lack or dysfunction is associated with severe health problems and thus the analysis of PMN physiology is a central issue. One prerequisite for PMN analysis is the availability of purified cells from primary organs. While human PMN are easily isolated from peripheral blood, this approach is less suitable for mice due to limited availability of blood. Instead, bone marrow (BM) is an easily available reservoir of murine PMN, but methods to obtain pure cells from BM are limited. We have developed a novel protocol allowing the isolation of highly pure untouched PMN from murine BM by negative immunomagnetic isolation using a complex antibody cocktail. The protocol is simple and fast (~1 h), has a high yield (5-10*10⁶ PMN per animal) and provides a purity of cells equivalent to positive selection (>80%). Most importantly, cells obtained by this method are non-activated and remain fully functional in vitro or after adoptive transfer into recipient animals. This method should thus greatly facilitate the study of primary murine PMN in vitro and in vivo.

Show MeSH
Related in: MedlinePlus