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Figure 4 | BMC Immunology

Figure 4

From: Age-associated alterations in CXCL1 chemokine expression by murine B cells

Figure 4

Ability of distinct splenic B cell subpopulations from young and aged mice to produce MIP-2 and KC chemokines. (A) Spleen cells were isolated from three young and aged mice, and then stained with anti-IgM, anti-CD23 and anti-CD21 Abs. Subsequently, NF, FO and MZ B cell subpopulations were sorted from IgM+ gated cell population. (B) Amounts of MIP-2 protein in supernatants of total IgM+ B cells and distinct splenic B cell subpopulations cultured in EL-4 system. NF, FO and MZ B cells within the respective gates shown were directly sorted into individual wells of 96 well plates (1,000 cells/well). Each well had 200 μl of medium containing irradiated EL-4 cells, LPS and macrophage supernatant. After 5 days of culture, supernatants were collected and analyzed by ELISA for MIP-2 and KC secretion. These values were significantly different from those in aged mice (* P < 0.05; ** P < 0.01; *** P < 0.005). (C) Real time RT-PCR analysis of MIP-2 and KC mRNA expression in 5 days-cultured B cells from young and aged mice. MIP-2 and KC mRNA levels in total IgM+, NF, FO and MZ B cells were measured by real time RT-PCR and normalized to threshold cycle (C t ) values of the co-amplified housekeeping gene GAPDH. Normalized values were calibrated to the value derived from EL-4 only controls and expressed as fold induction of mRNA. One representative experiment out of two is shown. These values were significantly different from those in aged mice (* P < 0.05; ** P < 0.01; *** P < 0.005).

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