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Cell density during differentiation can alter the phenotype of bone marrow-derived macrophages.

Lee CM, Hu J - Cell Biosci (2013)

Bottom Line: However, despite numerous protocols that are currently available, lack of a notable consensus on generating BMDMs may obscure the reliability in comparing findings from different studies or laboratories.With reference to previously published methods, bone marrow cells from wild type C57BL/6 mice were plated at either 4 × 10(5) cells or 5 × 10(6) cells per 10 cm and cultured in 20% L-cell conditioned media for 7 days, after which they were analyzed for cell surface markers, production of proinflammatory cytokines, and responsiveness to polarizing signals.BMDMs derived from higher plating density also secreted less proinflammatory cytokines such as IL-6, IL-12 and TNF-α and were less phagocytic, and had a different pattern of expression for M1- and M2-related genes upon LPS or IL-4 stimulation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Physiology and Experimental Medicine, SickKids, 555 University Avenue, M5G 1X8, Toronto, Ontario, Canada ; Laboratory Medicine & Pathobiology, University of Toronto, 1 King's College Circle, M5S 1A8, Toronto, Ontario, Canada.

ABSTRACT

Background: Bone marrow-derived macrophages (BMDMs) are widely used primary cells for studying macrophage function. However, despite numerous protocols that are currently available, lack of a notable consensus on generating BMDMs may obscure the reliability in comparing findings from different studies or laboratories.

Findings: In this study, we addressed the effect of cell density on the resulting macrophage population. With reference to previously published methods, bone marrow cells from wild type C57BL/6 mice were plated at either 4 × 10(5) cells or 5 × 10(6) cells per 10 cm and cultured in 20% L-cell conditioned media for 7 days, after which they were analyzed for cell surface markers, production of proinflammatory cytokines, and responsiveness to polarizing signals. Reproducibly, cells plated at lower density gave a pure population of CD11b(+)F4/80(+) macrophages (97.28 ± 0.52%) with majority being Ly-6C(-)Ly-6G(-) and c-Fms(+), while those plated at higher density produced less CD11b(+)F4/80(+) cells and a considerably higher proportion of CD11b(+)F4/80(+)CD11c(+) (68.72 ± 2.52%) and Ly-6C(-)Ly-6G(+) (71.10 ± 0.90%) cells. BMDMs derived from higher plating density also secreted less proinflammatory cytokines such as IL-6, IL-12 and TNF-α and were less phagocytic, and had a different pattern of expression for M1- and M2-related genes upon LPS or IL-4 stimulation.

Conclusions: Overall, our findings indicate that altering cell density during BMDM differentiation can give rise to distinct macrophage populations that could vary the outcome of a functional study.

No MeSH data available.


Related in: MedlinePlus

Differentiation of bone marrow cells at different densities results in phenotypically distinct BMDM populations. Plating cells at higher density produces (a) smaller and rounder macrophages, with top panel showing the forward-side scatter plot bottom panel showing the phase contrast images; (b) less CD11b+F4/80+ cells but more CD11b+F4/80+CD11c+; (c) less CD11b + c-Fms+; and (d) more CD11b+Ly-6G+ cells. For flow cytometric analysis, 2 × 105 BMDM cells detached from culture dishes by 5 mM EDTA 1× PBS were stained with appropriate conjugated primary antibodies, using unstained cells and isotype antibodies as negative controls and Fixable Violet™ staining (Invitrogen) for dead cell exclusion. Data was acquired by BD LSRII flow cytometer and analyzed with Flowjo software. Results represent the mean (±SE) of three experiments of three independent samples each, and statistical analysis was done by student t test with Welch’s correction where appropriate. *** P < 0.0005; ** P < 0.005; * P < 0.05.
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Figure 1: Differentiation of bone marrow cells at different densities results in phenotypically distinct BMDM populations. Plating cells at higher density produces (a) smaller and rounder macrophages, with top panel showing the forward-side scatter plot bottom panel showing the phase contrast images; (b) less CD11b+F4/80+ cells but more CD11b+F4/80+CD11c+; (c) less CD11b + c-Fms+; and (d) more CD11b+Ly-6G+ cells. For flow cytometric analysis, 2 × 105 BMDM cells detached from culture dishes by 5 mM EDTA 1× PBS were stained with appropriate conjugated primary antibodies, using unstained cells and isotype antibodies as negative controls and Fixable Violet™ staining (Invitrogen) for dead cell exclusion. Data was acquired by BD LSRII flow cytometer and analyzed with Flowjo software. Results represent the mean (±SE) of three experiments of three independent samples each, and statistical analysis was done by student t test with Welch’s correction where appropriate. *** P < 0.0005; ** P < 0.005; * P < 0.05.

Mentions: Flow cytometric analysis of monocytic/macrophage maturation markers of BMDMs generated under low (4 × 105) or high (5 × 106) plating concentrations revealed that the same bone marrow cells grown under different densities can give rise to phenotypically distinct populations (Figure 1). Reproducibly, cells plated at lower density gave a pure population of CD11b+F4/80+ macrophages (97.28 ± 0.52%) (Figure 1b) with majority being Ly-6C-Ly-6G- (60.46 ± 2.88%) and c-Fms+ (86.70 ± 2.95%) (Figure 1c, d) which denotes a mature phenotype[8], while those plated at higher density produced less CD11b+F4/80+ (89.18 ± 1.14%) cells and a considerably higher proportion of CD11c+ (68.72 ± 2.52%) and Ly-6C-Ly-6G+ (71.10 ± 0.90%) cells (Figure 1b,d).


Cell density during differentiation can alter the phenotype of bone marrow-derived macrophages.

Lee CM, Hu J - Cell Biosci (2013)

Differentiation of bone marrow cells at different densities results in phenotypically distinct BMDM populations. Plating cells at higher density produces (a) smaller and rounder macrophages, with top panel showing the forward-side scatter plot bottom panel showing the phase contrast images; (b) less CD11b+F4/80+ cells but more CD11b+F4/80+CD11c+; (c) less CD11b + c-Fms+; and (d) more CD11b+Ly-6G+ cells. For flow cytometric analysis, 2 × 105 BMDM cells detached from culture dishes by 5 mM EDTA 1× PBS were stained with appropriate conjugated primary antibodies, using unstained cells and isotype antibodies as negative controls and Fixable Violet™ staining (Invitrogen) for dead cell exclusion. Data was acquired by BD LSRII flow cytometer and analyzed with Flowjo software. Results represent the mean (±SE) of three experiments of three independent samples each, and statistical analysis was done by student t test with Welch’s correction where appropriate. *** P < 0.0005; ** P < 0.005; * P < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750618&req=5

Figure 1: Differentiation of bone marrow cells at different densities results in phenotypically distinct BMDM populations. Plating cells at higher density produces (a) smaller and rounder macrophages, with top panel showing the forward-side scatter plot bottom panel showing the phase contrast images; (b) less CD11b+F4/80+ cells but more CD11b+F4/80+CD11c+; (c) less CD11b + c-Fms+; and (d) more CD11b+Ly-6G+ cells. For flow cytometric analysis, 2 × 105 BMDM cells detached from culture dishes by 5 mM EDTA 1× PBS were stained with appropriate conjugated primary antibodies, using unstained cells and isotype antibodies as negative controls and Fixable Violet™ staining (Invitrogen) for dead cell exclusion. Data was acquired by BD LSRII flow cytometer and analyzed with Flowjo software. Results represent the mean (±SE) of three experiments of three independent samples each, and statistical analysis was done by student t test with Welch’s correction where appropriate. *** P < 0.0005; ** P < 0.005; * P < 0.05.
Mentions: Flow cytometric analysis of monocytic/macrophage maturation markers of BMDMs generated under low (4 × 105) or high (5 × 106) plating concentrations revealed that the same bone marrow cells grown under different densities can give rise to phenotypically distinct populations (Figure 1). Reproducibly, cells plated at lower density gave a pure population of CD11b+F4/80+ macrophages (97.28 ± 0.52%) (Figure 1b) with majority being Ly-6C-Ly-6G- (60.46 ± 2.88%) and c-Fms+ (86.70 ± 2.95%) (Figure 1c, d) which denotes a mature phenotype[8], while those plated at higher density produced less CD11b+F4/80+ (89.18 ± 1.14%) cells and a considerably higher proportion of CD11c+ (68.72 ± 2.52%) and Ly-6C-Ly-6G+ (71.10 ± 0.90%) cells (Figure 1b,d).

Bottom Line: However, despite numerous protocols that are currently available, lack of a notable consensus on generating BMDMs may obscure the reliability in comparing findings from different studies or laboratories.With reference to previously published methods, bone marrow cells from wild type C57BL/6 mice were plated at either 4 × 10(5) cells or 5 × 10(6) cells per 10 cm and cultured in 20% L-cell conditioned media for 7 days, after which they were analyzed for cell surface markers, production of proinflammatory cytokines, and responsiveness to polarizing signals.BMDMs derived from higher plating density also secreted less proinflammatory cytokines such as IL-6, IL-12 and TNF-α and were less phagocytic, and had a different pattern of expression for M1- and M2-related genes upon LPS or IL-4 stimulation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Physiology and Experimental Medicine, SickKids, 555 University Avenue, M5G 1X8, Toronto, Ontario, Canada ; Laboratory Medicine & Pathobiology, University of Toronto, 1 King's College Circle, M5S 1A8, Toronto, Ontario, Canada.

ABSTRACT

Background: Bone marrow-derived macrophages (BMDMs) are widely used primary cells for studying macrophage function. However, despite numerous protocols that are currently available, lack of a notable consensus on generating BMDMs may obscure the reliability in comparing findings from different studies or laboratories.

Findings: In this study, we addressed the effect of cell density on the resulting macrophage population. With reference to previously published methods, bone marrow cells from wild type C57BL/6 mice were plated at either 4 × 10(5) cells or 5 × 10(6) cells per 10 cm and cultured in 20% L-cell conditioned media for 7 days, after which they were analyzed for cell surface markers, production of proinflammatory cytokines, and responsiveness to polarizing signals. Reproducibly, cells plated at lower density gave a pure population of CD11b(+)F4/80(+) macrophages (97.28 ± 0.52%) with majority being Ly-6C(-)Ly-6G(-) and c-Fms(+), while those plated at higher density produced less CD11b(+)F4/80(+) cells and a considerably higher proportion of CD11b(+)F4/80(+)CD11c(+) (68.72 ± 2.52%) and Ly-6C(-)Ly-6G(+) (71.10 ± 0.90%) cells. BMDMs derived from higher plating density also secreted less proinflammatory cytokines such as IL-6, IL-12 and TNF-α and were less phagocytic, and had a different pattern of expression for M1- and M2-related genes upon LPS or IL-4 stimulation.

Conclusions: Overall, our findings indicate that altering cell density during BMDM differentiation can give rise to distinct macrophage populations that could vary the outcome of a functional study.

No MeSH data available.


Related in: MedlinePlus