Figure 2

Effect of aging on LPS-induced B cell proliferation and MIP-2 and KC production by splenic B cells. (A) In vitro proliferation of splenic B cells stimulated with LPS. Purified splenic B cells (1.25 × 10⁵/ml) were cultured with or without LPS. Proliferation was measured by [³H] thymidine uptake after 24 and 72 h of culture. Data are means ± SD of three mice in each group. The value was significantly different from that of aged mice. (** P < 0.01) (B) Splenic B cells from three to five young and aged mice were stimulated with 10 μg/ml of LPS for 4 and 24 h. After stimulation, cell-free supernatants were collected and assayed by ELISA for MIP-2 and KC secretion. One representative experiment out of three is shown. (C) 24 h LPS-stimulated splenic B cells were subjected to immunofluorescence staining with anti-MIP-2 and anti-KC antibodies and DNA dye DAPI as described in Figure 1. One representative experiment out of three is shown. Control anti-goat IgG staining in both young and aged B cells are shown as inserts in this panel. (D) After stimulation, cells were harvested, and RNA was prepared for MIP-2 and KC specific RT-PCR. The housekeeping gene β-actin was amplified as an internal control. Data shown are representative of two independent experiments. (E) MIP-2 and KC mRNA levels were measured by real time RT-PCR and normalized to threshold cycle (Ct) values of the co-amplified house-keeping gene GAPDH. Normalized values were calibrated to the value derived from non-stimulated controls and shown as fold change of mRNA expression. Data shown are representative of two independent experiments. Value were significantly different from those in aged mice (** P < 0.01; *** P < 0.005)