RNA was prepared from each sort-purified B cell subset and reverse transcribed

RNA was prepared from each sort-purified B cell subset and reverse transcribed. in C57BL/6 B cells. Peritoneal washout cells and spleen cells were obtained from 3-month-old C57BL/6J mice, immunofluorescently stained, and sorted for peritoneal B-1a (B220loCD5+), CD25+ B-1a (B220loCD5+CD25+), CD25? B-1a (B220loCD5+CD25?), splenic B2 (B220+CD5?CD23+), and GC (B220+/GL-7+/PNAhigh) cells, as shown in Physique ?Physique1.1. RNA was prepared from each sort-purified B cell subset and reverse transcribed. The level of relative to 2-microglobulin was determined by real-time PCR (SYBR Green) with the primers described in Section Materials and Methods. The means of three impartial experiments cFMS-IN-2 are shown in (A), along with lines indicating SEMs. The level of relative to actin was determined by real-time PCR (Taqman) with the primers described in Section Materials and Methods. The means of three impartial experiments are shown in (B), along with lines indicating SEMs. image_2.tif (1.2M) GUID:?D9643765-C3C5-4C14-BCC4-F8F9B49EBAEE Abstract B-1a cells are innate-like B-lymphocytes producing natural antibodies. Activation-induced cytidine deaminase (AID), a product of the gene, plays a central role in class-switch recombination and somatic hypermutation in B cells. Although a role for in B-1a cells has been suggested on the basis of experiments with knock out (KO) mice, whether B-1a cells express expression is concentrated in the CD25+ B-1a cell subpopulation. These results suggest the possibility that previous studies of memory B cells identified on the basis of expression may have inadvertently included an unknown number of CD25+ B-1a cells. Although B-1a cells develop normally in the absence of and Gene Expression Is Restricted to the CD25+ B-1a Cell Subset The expression level of Vasp was evaluated in sort-purified peritoneal B-1a cells, peritoneal CD25+ B-1a cells (4), peritoneal CD25? B-1a cells, splenic B2 cells, and GC B cells from unmanipulated mice. The sorting strategy for isolating these populations is usually shown in Physique ?Figure1A.1A. GC B cells displayed a high level of expression, which is usually consistent with previous reports (12), in contrast to splenic B-2 cells that expressed very little than that by splenic B-2 cells, but less than that by GC B cells (Physique ?(Figure1B).1B). We then examined CD25+ B-1a cells in comparison to CD25? B-1a cells and found that CD25+ B-1a cells exhibited a higher level of expression than did CD25? B-1a cells, total B-1a cells, and splenic B-2 cells, although this was still less than the level expressed by GC B cells. These results were confirmed using Taqman primers and probe (Physique S1 in Supplementary Material). Peritoneal CD25+ B-1a cells from C57BL/6 mice were also found to express in greater amounts than that by CD25? B-1a cells (Physique S2 in Supplementary Material). The mean level of expression in BALB/c CD25+ B-1a cells was 18-fold more than that of splenic B-2 cells but 40-fold less than that of GC B cells. Thus, B-1a cells, especially CD25+ B-1a cells, express gene expression in B cFMS-IN-2 cells. Peritoneal washout cells and spleen cells were obtained from 3-month-old BALB/c-ByJ mice, immunofluorescently stained, and sorted for peritoneal B-1a (B220loCD5+), CD25+ B-1a (B220loCD5+CD25+), CD25? B-1a (B220loCD5+CD25?), splenic B2 (B220+CD5?CD23+), and germinal center (GC, B220+/GL-7+/PNAhigh) cells. The sorting strategy for these populations is usually shown in (A). RNA was prepared cFMS-IN-2 from each sort-purified B cell subset and reverse transcribed. (A) The level of relative to 2-microglobulin was determined by real-time PCR (SYBR Green) with the primers described in Section Materials and Methods. The means of three impartial experiments are shown in (B), along with lines indicating SEMs. The Number of CD25+ B-1a Cells Is usually Unchanged in AID KO Mice lacking the AID gene around the BALB/c background were assessed for numbers of total peritoneal cells, total peritoneal lymphocytes, B-1a cells, CD25+ B-1a cells, and CD25? B-1a cells. There was no significant difference in the total number of peritoneal lymphocytes in AID KO mice (4.3??106??0.71) compared to that in WT mice (3.0??106??0.17) (Physique ?(Figure2A),2A), although the total number of cells in the peritoneal cavities of AID KO mice was greater than the number in WT mice, presumably due to differences in a non-lymphoid population, such as myeloid cells. Next, the total numbers of B-1a, CD25+ B-1a, and CD25? B-1a cells were assessed in WT and AID KO mice. The results exhibited that there is no significant difference in the total numbers of peritoneal B-1a, CD25+ B-1a, or CD25? B-1a cells from AID KO mice compared to those in WT controls (Physique ?(Figure2B).2B). Thus, AID does not appear to be required for the cFMS-IN-2 development of early appearing CD25+ or CD25? B-1a cells. Open in a separate window Physique 2 Number of B-1a cells in activation-induced cFMS-IN-2 cytidine deaminase (AID) knock out (KO) mice. Peritoneal washout cells were obtained from 3-month-old wild-type (WT) and AID KO mice around the BALB/c-ByJ background. (A) The total number of peritoneal cells and the total number of lymphocytes (based on the lymphocyte gate) are shown. (B) Peritoneal washout cells were stained with anti-B220-pCP-Cy5.5, anti-CD5-Alexa 647, anti-CD25-PE, and.

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