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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.


Expression of macrophage polarization genes in BMDMs. (a) Expression of genes related to the M1 polarization state upon activation with 100 ng/mL LPS for 18 hours and (b) M2 polarization state after treatment with 10 ng/mL of IL-4 (Peprotech) for 18 hours. Total RNA was isolated by GE RNAspin mini kit and reverse transcribed with Super Script II (Invitrogen) and run on ABI 7500 qPCR machine using ABI SYBERGreen Mastermix. Statistical analysis was done by student t test with Welch’s correction where appropriate. Result is a representative of three independent experiments with three biological samples per datapoint. *** P < 0.0005; ** P < 0.005; * P < 0.05.
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Figure 3: Expression of macrophage polarization genes in BMDMs. (a) Expression of genes related to the M1 polarization state upon activation with 100 ng/mL LPS for 18 hours and (b) M2 polarization state after treatment with 10 ng/mL of IL-4 (Peprotech) for 18 hours. Total RNA was isolated by GE RNAspin mini kit and reverse transcribed with Super Script II (Invitrogen) and run on ABI 7500 qPCR machine using ABI SYBERGreen Mastermix. Statistical analysis was done by student t test with Welch’s correction where appropriate. Result is a representative of three independent experiments with three biological samples per datapoint. *** P < 0.0005; ** P < 0.005; * P < 0.05.

Mentions: To examine whether the observed phenotypic difference had a functional consequence, BMDMs grown under different densities were compared for their ability to secrete cytokines and phagocytose foreign materials. Noticeably, BMDMs derived from higher plating density secreted less proinflammatory cytokines such as TNF-α, IL-12, IL-6, KC and MIP-1α (Figure 2a) in response to LPS, were less phagocytic (Figure 2b), and had a different pattern of expression for M1- and M2-related genes upon LPS or IL-4 stimulation (Figure 3). It was interesting to note, however, that cells grown under higher plating density displayed increased nitric oxide (NO) production (Figure 2c), despite the fact that the same number of live cells (dead cells excluded by trypan blue staining) of each group were used for functional assays. In conclusion, our data indicate that BMDM density during in vitro differentiation can have a functional implication on the final macrophage populations, at least in BMDMs derived from C57BL/6 strain of mice.


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

Lee CM, Hu J - Cell Biosci (2013)

Expression of macrophage polarization genes in BMDMs. (a) Expression of genes related to the M1 polarization state upon activation with 100 ng/mL LPS for 18 hours and (b) M2 polarization state after treatment with 10 ng/mL of IL-4 (Peprotech) for 18 hours. Total RNA was isolated by GE RNAspin mini kit and reverse transcribed with Super Script II (Invitrogen) and run on ABI 7500 qPCR machine using ABI SYBERGreen Mastermix. Statistical analysis was done by student t test with Welch’s correction where appropriate. Result is a representative of three independent experiments with three biological samples per datapoint. *** 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 3: Expression of macrophage polarization genes in BMDMs. (a) Expression of genes related to the M1 polarization state upon activation with 100 ng/mL LPS for 18 hours and (b) M2 polarization state after treatment with 10 ng/mL of IL-4 (Peprotech) for 18 hours. Total RNA was isolated by GE RNAspin mini kit and reverse transcribed with Super Script II (Invitrogen) and run on ABI 7500 qPCR machine using ABI SYBERGreen Mastermix. Statistical analysis was done by student t test with Welch’s correction where appropriate. Result is a representative of three independent experiments with three biological samples per datapoint. *** P < 0.0005; ** P < 0.005; * P < 0.05.
Mentions: To examine whether the observed phenotypic difference had a functional consequence, BMDMs grown under different densities were compared for their ability to secrete cytokines and phagocytose foreign materials. Noticeably, BMDMs derived from higher plating density secreted less proinflammatory cytokines such as TNF-α, IL-12, IL-6, KC and MIP-1α (Figure 2a) in response to LPS, were less phagocytic (Figure 2b), and had a different pattern of expression for M1- and M2-related genes upon LPS or IL-4 stimulation (Figure 3). It was interesting to note, however, that cells grown under higher plating density displayed increased nitric oxide (NO) production (Figure 2c), despite the fact that the same number of live cells (dead cells excluded by trypan blue staining) of each group were used for functional assays. In conclusion, our data indicate that BMDM density during in vitro differentiation can have a functional implication on the final macrophage populations, at least in BMDMs derived from C57BL/6 strain of mice.

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.