COX-1 is required for early B-cell development. (A) Flow cytometric analysis of apoptosis (annexin V⁺) in B220⁺ B cells and CD3e⁺ T lymphocytes from BM in COX-1^−/− and WT mice. (Left) Representative from a single experiment. (Right) Mean ± SEMs from 4 independent experiments. (B) (Left) mRNA expression of COX-1 and COX-2 in distinct stages of developing B cells from mouse BM was evaluated by qRT-PCR. The subpopulations were purified by flow cytometric sorting based on the following surface markers: pre-pro-B (AA4.1⁺B220⁺CD19⁻CD24⁻), pro-B (B220⁺CD43⁺IgM⁻), pre-B(B220⁺CD43⁻IgM⁻), and immature B (B220⁺IgM⁺). (Right) Relative expression of COX-1 and other genes in sorted pro-B cells was determined by qRT-PCR. β-actin was used to normalize gene expression, the group with lowest expression was artificially set as 1. Mean ± SEMs of 3 independent experiments are shown. (C) Flow cytometry profiles of distinct B-cell compartments in BM from COX-1^−/− and WT mice. (D) BM cells were cocultured on OP9 stromal cells with IL-7 for 6 days (upper), or purified pro-B cells were cultured with IL-7, SCF, and Flt3L cytokines (lower), from COX-1^−/− and WT mice; the frequencies of pre-B cells generated were then determined by flow cytometric analysis. (E-G) BM transplantation: A 1:1 mixture of BM cells from COX-1^−/− or WT mice (CD45.2⁺) with BM from syngenic (CD45.1⁺) were injected into irradiated syngenic mice (5 × 10⁶ cells per mouse, n = 6). Mice were killed 6 weeks after transplantation. The levels of (E) total B cells and (F) developing B cells among CD45.2⁺ cells in recipients were determined by flow cytometry analysis. (G) Relative WT/knockout (KO) ratios of B-cell subpopulations in F were normalized against pre-pro-B cells. (C-G) (Upper) Representative from 1 single experiment; numbers in quadrants indicate percentage of total living cells. (Lower) Mean ± SEMs from (C,E-G) 6 mice or (D) 3 independent experiments. *P < .05 and **P < .01, using unpaired Student t tests.