Journal: PLoS ONE

Article Title: PP4 Is Essential for Germinal Center Formation and Class Switch Recombination in Mice

doi: 10.1371/journal.pone.0107505

Figure Lengend Snippet: ( A ) FACS assay of apoptosis of splenic B cells that were freshly isolated from WT and CD23 cre PP4 F/F mice (n = 3/group) and subjected to AnnexinV vs 7AAD staining. Viabilities are shown for the immature (IM), follicular (FO) and marginal zone (MZ) B cell populations. Numbers in quadrants represent the percentage of the indicated population relative to the total. ( B ) Quantitation of the percentage of viable (AnnexinV − 7AAD − ) cells in the IM, FO and MZ B cell populations in (A). Data are the mean ± SD of triplicate samples from each group. For (A–B), results are representative of two independent experiments. ( C ) In vivo BrdU incorporation assay. Left panel: WT and CD23 cre PP4 F/F mice (n = 3/group) were i.p. injected with BrdU twice daily (8 h apart) on 3 consecutive days. IM, FO and MZ splenocytes were freshly sorted, subjected to intracellular staining with anti-BrdU plus 7AAD, and analyzed by FACS. Right panel: Diagram indicating relative position of specific mitotic phase cells in the FACS profiles in the left panel. ( D ) Quantitation of the results in (C) showing the percentages of cells in the G0/G1, S and G2/M phases. Data are the mean ± SD of triplicate samples from each group. For (C–D), results are representative of two independent experiments. ( E ) In vitro BrdU incorporation assay. Splenic B cells were freshly isolated from WT (n = 3) or CD23 cre PP4 F/F (n = 4) mice and cultured in BrdU-containing maintenance medium alone (Medium), or in maintenance medium containing 1 µg/ml anti-IgM (IgM low), 10 µg/ml anti-IgM (IgM high), 5 µg/ml anti-CD40 Ab (CD40), 1 µg/ml anti-IgM plus anti-CD40 Abs (IgM/CD40), 1 µg/ml LPS (LPS low), or 10 µg/ml LPS (LPS high). After 48 h, cells were analyzed by FACS. Data shown are the quantitation of the mean percentage ± SD of B cells in S phase relative to total B cells after 48 h stimulation. ( F ) Quantitation of the mean percentage ± SD of B cells in G2/M phase from the data in (E) after 48 h stimulation. ( G ) Quantitation of the mean percentage ± SD of activated (CD80 + CD86 + ) B cells from the data in (E) after 24 h (d1) or 48 h (d2) of stimulation. ( H ) Quantitation of the mean percentage ± SD of CD40 + CD25 + B cells from the data in (G) after 48 h (d2) stimulation. For (E–H), results are representative of two independent experiments.

Article Snippet: Purified splenic B cells were seeded at a density of 1×10 6 /ml and cultured for 4 days in RPMI medium containing either 50 μg/ml lipopolysaccharide (LPS; InvivoGen) to induce switching from IgM to IgG 3 , or in RPMI medium containing 50 μg/ml LPS plus 10 ng/ml IL-4 (PeproTech) to induce switching from IgM to IgG 1 .

Techniques: Isolation, Staining, Quantitation Assay, In Vivo, BrdU Incorporation Assay, Injection, In Vitro, Cell Culture

Journal: PLoS ONE

Article Title: PP4 Is Essential for Germinal Center Formation and Class Switch Recombination in Mice

doi: 10.1371/journal.pone.0107505

Figure Lengend Snippet: ( A ) FACS profiles of IgG 1 vs IgG 3 expression by B220 + IgM − IgD − gated B cells that were isolated from WT and CD23 cre PP4 F/F mice (n = 3/group) and stimulated in vitro with LPS for 4 days. ( B ) Quantitation of the percentage of IgG 3 + -switched B cells among total B cells from the data in (A). Data are the mean ± SD of triplicates. For (A–B), results are representative of two independent experiments. ( C ) FACS profiles of IgG 1 vs IgG 3 expression by B220 + IgM − IgD − -gated B cells that were isolated from WT and CD23 cre PP4 F/F mice (n = 3/group) and stimulated in vitro with LPS plus IL-4 for 4 days. Data were analyzed as for (A). ( D ) Quantitation of the percentage of IgG 1 + -switched B cells among total B cells from the data in (C). For (C–D), results are representative of two independent experiments. ( E ) RT-PCR analysis of mRNA from B cells that were isolated from WT and CD23 cre PP4 F/F mice (n = 4/group), and either left unstimulated or stimulated in vitro with LPS or LPS plus IL-4 for 4 days. Samples were analyzed to detect AID and the indicated switched transcripts. HPRT, loading control. ( F ) RT-PCR analysis of the indicated germline transcripts in the B cells in (E). Loading control was the same as in (E). For (E–F), results are representative of two independent experiments. ( G ) DC-PCR analysis of genomic DNA prepared from resting B cells isolated from WT and CD23 cre PP4 F/F mice (n = 4/group). Genomic DNA was digested with Eco RI, ligated and utilized as template for PCR to detect the recombined Sµ-Sγ3 sequence (see ). nAChRe, loading control. ( H ) DC-PCR analysis of genomic DNA prepared from WT and CD23 cre PP4 F/F B cells that were stimulated in vitro with LPS plus IL-4 for 4 days. The recombined Sµ-Sγ1 sequence was detected as illustrated in . For (G–H), results are representative of two independent experiments.

Article Snippet: Purified splenic B cells were seeded at a density of 1×10 6 /ml and cultured for 4 days in RPMI medium containing either 50 μg/ml lipopolysaccharide (LPS; InvivoGen) to induce switching from IgM to IgG 3 , or in RPMI medium containing 50 μg/ml LPS plus 10 ng/ml IL-4 (PeproTech) to induce switching from IgM to IgG 1 .

Techniques: Expressing, Isolation, In Vitro, Quantitation Assay, Reverse Transcription Polymerase Chain Reaction, Control, Sequencing

Journal: PLoS ONE

Article Title: PP4 Is Essential for Germinal Center Formation and Class Switch Recombination in Mice

doi: 10.1371/journal.pone.0107505

Figure Lengend Snippet: ( A ) Western blot (WB) analysis of splenic B cells that were isolated from WT and CD23 cre PP4 F/F mice (n = 6/group) and left unstimulated (0), or stimulated in vitro with anti-CD40 Ab for the indicated times. Lysates were subjected to WB to detect p-Erk1/2 (T202/Y204), total Erk, p-JNK (T183/Y185), total JNK, IκBα, p-Akt/PKB (S473), and total Akt. Results are representative of three independent experiments. ( B ) WB analysis of splenic B cells that were isolated from WT and CD23 cre PP4 F/F mice (n = 6/group) and left unstimulated (0), or stimulated with anti-IgM Ab for the indicated times. Lysates were analyzed as in (A). Results are representative of two independent experiments. ( C ) FACS profile of in vitro BrdU incorporation assay. Splenic B cells were freshly isolated from BCR HEL /CD23 cre PP4 +/+ (n = 3) and BCR HEL /CD23 cre PP4 F/F (n = 3) mice and cultured in BrdU-containing maintenance medium alone (Medium), or in maintenance medium containing 1 µg/ml HEL, 1 µg/ml anti-IgM plus anti-CD40 Abs (IgM/CD40) and 0.5 µg/ml LPS (LPS low). After 72 h, cells were analyzed by FACS. Numbers in upper quadrants represent the percentage of B cells in S phase relative to the total B cells after 72 h stimulation, while numbers in left quadrants represent the percentage of apoptotic B cells. ( D ) Quantitation of the mean percentage ± SD of B cells in S phase from the data in (C). β-tubulin, loading control. Results are representative of two independent experiments. ( E ) FACS profile of in vivo BrdU incorporation assay. BCR HEL /CD23 cre PP4 +/+ (n = 4) and BCR HEL /CD23 cre PP4 F/F (n = 4) mice were immunized with HEL in alum at day 0 and injected with BrdU from day 3 in 4 consecutive days as illustrated in . At day 7 after immunization, splenic B cells were harvested and analyzed by FACS. Numbers in quadrants represent the percentage of B cells in S phase (BrdU + ) relative to total B cells. Results are representative of two independent experiments. ( F ) Quantitation of the mean percentage ± SD of B cells in S phase from the data in (E).

Article Snippet: Purified splenic B cells were seeded at a density of 1×10 6 /ml and cultured for 4 days in RPMI medium containing either 50 μg/ml lipopolysaccharide (LPS; InvivoGen) to induce switching from IgM to IgG 3 , or in RPMI medium containing 50 μg/ml LPS plus 10 ng/ml IL-4 (PeproTech) to induce switching from IgM to IgG 1 .

Techniques: Western Blot, Isolation, In Vitro, BrdU Incorporation Assay, Cell Culture, Quantitation Assay, Control, In Vivo, Injection