Journal:

Article Title: Human blood IgM "memory" B cells are circulating splenic marginal zone B cells harboring a prediversified immunoglobulin repertoire

doi: 10.1182/blood-2004-01-0346

Figure Lengend Snippet: Purified B cells from blood and spleen are analyzed separately for IgM, CD21, CD23 and CDlc surface expression after gating of the three different CD19-positive lymphocyte subsets distinguished by IgD and CD27 labelling. These data correspond to one representative case out of 4 different individuals. Naive B cells (IgD+CD27−), bold line; IgD+CD27+ B cells, grey shadow; IgD−CD27+, thin line. Percentages of cells in the two CD27+ quadrants are indicated. The absence of IgM-positive cells among the IgD−CD27+ subset is noticeable.

Article Snippet: The following antibodies coupled with biotin, fluorescein isothiocyanate (FTTC), R-phycoerythrin (PE), allophycocyanin (APC), Cy-Chrome ™ (Cy) or with the tandem dye PE-Cyanin 5.1 (PC5) were used for flow cytometry or cell sorting: PC5-anti-CD19 (clone J4.119) and PE-anti-CD27 (clone 1A4-CD27) from Beckman Coulter (Fullerton, CA); APC-anti-CD19 (clone HIB19), Cy-anti-CD21 (clone B-Ly4), FTTC-anti-CD27 (clone M-T271), PE-anti-CD23 (clone M-L233) and biotin anti-IgD (clone IA6-2) from BD-Pharmingen (San Jose, CA); goat anti-human IgD-FITC and biotinylated goat F(ab′) 2 anti-human IgM from Caltag (Burlingame, CA).

Techniques: Purification, Expressing

Journal:

Article Title: Human blood IgM "memory" B cells are circulating splenic marginal zone B cells harboring a prediversified immunoglobulin repertoire

doi: 10.1182/blood-2004-01-0346

Figure Lengend Snippet: Serial cryosections of an adult human spleen are stained with anti-CD20, anti-IgD, anti-CD 1c and anti-CD27 antibodies (ABC technique; Original magnification: 25X) Marginal zone B cells are IgDlow CD27+CDlchigh. Note the more intense staining for IgD of the corona (Co) compared to the marginal zone B cells (MZ) while the reverse is true for CDlc. The intense IgD staining of outer marginal zone B cells has been described previousl(16). GC, germinal center.

Article Snippet: The following antibodies coupled with biotin, fluorescein isothiocyanate (FTTC), R-phycoerythrin (PE), allophycocyanin (APC), Cy-Chrome ™ (Cy) or with the tandem dye PE-Cyanin 5.1 (PC5) were used for flow cytometry or cell sorting: PC5-anti-CD19 (clone J4.119) and PE-anti-CD27 (clone 1A4-CD27) from Beckman Coulter (Fullerton, CA); APC-anti-CD19 (clone HIB19), Cy-anti-CD21 (clone B-Ly4), FTTC-anti-CD27 (clone M-T271), PE-anti-CD23 (clone M-L233) and biotin anti-IgD (clone IA6-2) from BD-Pharmingen (San Jose, CA); goat anti-human IgD-FITC and biotinylated goat F(ab′) 2 anti-human IgM from Caltag (Burlingame, CA).

Techniques: Staining

Journal:

Article Title: Human blood IgM "memory" B cells are circulating splenic marginal zone B cells harboring a prediversified immunoglobulin repertoire

doi: 10.1182/blood-2004-01-0346

Figure Lengend Snippet: Patients L.D. and C.A. (patient one) have been reported previously (20,21). Control is a eleven-year-old child, age-matched with patient C.A. Since IgD+CD27+cells co-express IgM, the IgM+IgD+CD27+ subset is analyzed after IgD, CD27 and CD19 labeling of purified B cells. IgD and CD27 expression is shown after gating on CD19-positive cells. Percentages of cells in the naive and the two CD27+ quadrants are indicated.

Article Snippet: The following antibodies coupled with biotin, fluorescein isothiocyanate (FTTC), R-phycoerythrin (PE), allophycocyanin (APC), Cy-Chrome ™ (Cy) or with the tandem dye PE-Cyanin 5.1 (PC5) were used for flow cytometry or cell sorting: PC5-anti-CD19 (clone J4.119) and PE-anti-CD27 (clone 1A4-CD27) from Beckman Coulter (Fullerton, CA); APC-anti-CD19 (clone HIB19), Cy-anti-CD21 (clone B-Ly4), FTTC-anti-CD27 (clone M-T271), PE-anti-CD23 (clone M-L233) and biotin anti-IgD (clone IA6-2) from BD-Pharmingen (San Jose, CA); goat anti-human IgD-FITC and biotinylated goat F(ab′) 2 anti-human IgM from Caltag (Burlingame, CA).

Techniques: Labeling, Purification, Expressing

Journal:

Article Title: Human blood IgM "memory" B cells are circulating splenic marginal zone B cells harboring a prediversified immunoglobulin repertoire

doi: 10.1182/blood-2004-01-0346

Figure Lengend Snippet: Each column represents microarray data from a sample of the indicated cell subtype and each row represents the expression of a single gene. The spleen IgD+CD27+ and IgD−CD27+ populations are obtained from two separate donors, with one of the two samples prepared in duplicate. Red squares indicate increased expression and green squares indicate decreased expression relative to the median expression of the gene according to the color bar shown. Gray squares indicate missing or excluded data, a) The array dendrogram obtained by clustering the 49 genes that differentiated (p<0.005, two-fold higher expression) the splenic IgD+CD27+ samples from the splenic IgD−CD27+ samples. The red branches indicate the co-clustering of the blood IgD+CD27+ samples with the splenic IgD+CD27+ samples and the blue branches indicate the co-clustering of the blood IgD−CD27+ samples with their respective splenic IgD−CD27+ samples, b) The 37 genes that achieved statistical significance with a two-fold higher mean expression when comparing the IgD+CD27+ cell populations with the memory IgD−CD27+ cell populations.

Article Snippet: The following antibodies coupled with biotin, fluorescein isothiocyanate (FTTC), R-phycoerythrin (PE), allophycocyanin (APC), Cy-Chrome ™ (Cy) or with the tandem dye PE-Cyanin 5.1 (PC5) were used for flow cytometry or cell sorting: PC5-anti-CD19 (clone J4.119) and PE-anti-CD27 (clone 1A4-CD27) from Beckman Coulter (Fullerton, CA); APC-anti-CD19 (clone HIB19), Cy-anti-CD21 (clone B-Ly4), FTTC-anti-CD27 (clone M-T271), PE-anti-CD23 (clone M-L233) and biotin anti-IgD (clone IA6-2) from BD-Pharmingen (San Jose, CA); goat anti-human IgD-FITC and biotinylated goat F(ab′) 2 anti-human IgM from Caltag (Burlingame, CA).

Techniques: Microarray, Expressing

Journal:

Article Title: Human blood IgM "memory" B cells are circulating splenic marginal zone B cells harboring a prediversified immunoglobulin repertoire

doi: 10.1182/blood-2004-01-0346

Figure Lengend Snippet: Amplification of V3-15-Cμ mRNA sequences was performed from naive and IgM+IgD+CD27+ B cells of a 9-year old child undergoing splenectomy and immunized against Streptococcus pneumoniae and Neisseria meningitidis (plain polysaccharidic vaccines). The following samples were analyzed: blood before immunization, blood at the time of splenectomy (i.e. 8 days after immunization), spleen, blood 5 weeks after immunization. The first V3-15-specific PCR products were further amplified with V3-15-specific FR3 and C7μ primers, and the resulting products fractionated by denaturing gel electrophoresis. A specific CDR3 size was excised from the gel after silver staining and reamplified with the same FR3 and Cμ primers, and sequences determined after cloning. Several PCR amplifications were performed from two independent cDNAs for each cell sample. The recurrent CDR3s encompassing the V3-15 and JH3 junctions observed in the various IgM+IgD+CD27+ fractions are shown, with the most frequently occurring sequence taken as reference (CDR3 is defined as amino acids included between the conserved Cys residue of FR3 and Trp residue of JH segments). The asterisk (*) marks clones found repeatedly in independent PCR amplifications.

Article Snippet: The following antibodies coupled with biotin, fluorescein isothiocyanate (FTTC), R-phycoerythrin (PE), allophycocyanin (APC), Cy-Chrome ™ (Cy) or with the tandem dye PE-Cyanin 5.1 (PC5) were used for flow cytometry or cell sorting: PC5-anti-CD19 (clone J4.119) and PE-anti-CD27 (clone 1A4-CD27) from Beckman Coulter (Fullerton, CA); APC-anti-CD19 (clone HIB19), Cy-anti-CD21 (clone B-Ly4), FTTC-anti-CD27 (clone M-T271), PE-anti-CD23 (clone M-L233) and biotin anti-IgD (clone IA6-2) from BD-Pharmingen (San Jose, CA); goat anti-human IgD-FITC and biotinylated goat F(ab′) 2 anti-human IgM from Caltag (Burlingame, CA).

Techniques: Amplification, Nucleic Acid Electrophoresis, Silver Staining, Clone Assay, Sequencing

Journal:

Article Title: Human blood IgM "memory" B cells are circulating splenic marginal zone B cells harboring a prediversified immunoglobulin repertoire

doi: 10.1182/blood-2004-01-0346

Figure Lengend Snippet: A, C. Percentage of IgM+IgD+CD27+ peripheral B cells from normal (A) and asplenic children (C) below five years.

Article Snippet: The following antibodies coupled with biotin, fluorescein isothiocyanate (FTTC), R-phycoerythrin (PE), allophycocyanin (APC), Cy-Chrome ™ (Cy) or with the tandem dye PE-Cyanin 5.1 (PC5) were used for flow cytometry or cell sorting: PC5-anti-CD19 (clone J4.119) and PE-anti-CD27 (clone 1A4-CD27) from Beckman Coulter (Fullerton, CA); APC-anti-CD19 (clone HIB19), Cy-anti-CD21 (clone B-Ly4), FTTC-anti-CD27 (clone M-T271), PE-anti-CD23 (clone M-L233) and biotin anti-IgD (clone IA6-2) from BD-Pharmingen (San Jose, CA); goat anti-human IgD-FITC and biotinylated goat F(ab′) 2 anti-human IgM from Caltag (Burlingame, CA).

Techniques: