Journal: Stem Cell Research & Therapy

Article Title: Isolation and characterization of equine native MSC populations

doi: 10.1186/s13287-017-0525-2

Figure Lengend Snippet: Isolation of CD34 + and CD146 + cells from adipose SVF. a , b Events displayed as FSC-A vs FSC-H to select singlets ( a ) were further gated to exclude cell fragments or noncellular material ( b ). c – e Sequence of plots showing selection of DAPI-negative, CD144(AF405)-negative, and CD45(FITC)-negative cells to obtain live, endothelial-negative, and hematopoietic-negative fractions, respectively. ( c1–c4 ) Dot-plots for CD144 ( c1 , c2 ) and CD45 ( c3 , c4 ) controls showing unstained cells ( c1 , c3 ) and secondary antibody conjugated to AF405 ( c2 ) or isotype conjugated to FITC ( c4 ). f Double-plot displaying CD34 + (Q1) and CD146 + (Q4) cell subpopulations and respective isotype controls ( c5 ) conjugated to PE and AF647, respectively. Filters used: 450/50 for AF405, 525/50 for FITC, 586/15 for PE, and 670/30 for AF647. FSC-A forward scatter area, FSC-H forward scatter height, SSC-A side scatter area

Article Snippet: Isotype controls used (raised in mouse) were IgG1-FITC (MCA928F; AbD serotec-BioRad), IgG1-AF647 (MCA928A647, AbD serotec-BioRad), and IgG1-PE (21275514; ImmunoTools).

Techniques: Isolation, Sequencing, Selection

Journal: eLife

Article Title: Protein kinase Cδ is essential for the IgG response against T-cell-independent type 2 antigens and commensal bacteria

doi: 10.7554/eLife.72116

Figure Lengend Snippet: ( A and B ) Naive B cells purified from the spleen of Igk −/− B1-8 flox/+ mice were stimulated with NP-CGG or NP-Ficoll. ( A ) 3 H-thymidine incorporation on day 3. ( B ) IgM and IgG concentrations in the culture supernatants on day 3. ( C–E ) Igk −/− B1-8 flox/+ B cells were stimulated with none (–), NP-CGG, or NP-Ficoll and IL-1α, IL-1β, or IFNα, as indicated. ( C ) Enzyme-linked immunosorbent assay (ELISA) of IgG3 in the culture supernatant on day 3. AU, arbitrary units. ( D ) Representative flow cytometric plots on day 3 with the numbers indicating percentages of IgG3 + B cells (top). The frequencies of IgG3 + cells among the B cells (bottom). ( E ) quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analysis of the circle Iγ3-Cµ and Aicda transcripts on day 2. Data are means ± standard deviations (SDs) of two ( B and C ), two to three ( D ), or three ( A ) biological replicates or three technical replicates ( E ). The data are representative of at least three ( A and B ) or two ( C–E ) independent experiments. *p < 0.05; ***p < 0.001; ****p < 0.0001; p values were calculated by one- ( B ) or two ( C and D )-way analysis of variance (ANOVA) with Tukey’s test. Figure 1—source data 1. Source data for .

Article Snippet: Antibody , Goat F(ab')2 Anti-Mouse IgG2b, Human ads-FITC (Goat Polyclonal) , Southern Biotech , Cat# 1092-02 , Flow cytometry (1/300).

Techniques: Purification, Enzyme-linked Immunosorbent Assay, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR

Journal: eLife

Article Title: Protein kinase Cδ is essential for the IgG response against T-cell-independent type 2 antigens and commensal bacteria

doi: 10.7554/eLife.72116

Figure Lengend Snippet: ( A–C ) Igk −/− B1-8 flox/+ naive B cells were stimulated with nothing (–), NP-CGG, or NP-Ficoll in the presence of LPS, R-848, or CpG. ( A ) IgG concentration in the culture supernatants on day 3. ( B ) Representative flow cytometric plots on day 3 showing percentages of IgG + B cells. ( C ) qRT-PCR analysis of Aicda transcripts on day 2. ( D and E ) Igk −/− B1-8 flox/+ naive B cells were stimulated with NP-Ficoll and each indicated cytokine. ( D ) IgG3 in the culture supernatants on day 4 titrated by enzyme-linked immunosorbent assay (ELISA). AU, arbitrary units. ( E ) Representative flow cytometric profiles of IgG1, IgG2b, IgG2c, and IgG3 on day 3 (left) and the ratio of B cells expressing these IgG subclasses estimated from the flow cytometry data (right). Data are means of three biological replicates ( E ), means ± standard deviations (SDs) of two biological replicates ( A ), or three technical replicates ( C ). The data are representative of two independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; p values were calculated by two-way analysis of variance (ANOVA) with Tukey’s test ( A ). Figure 1—figure supplement 1—source data 1. Source data for .

Article Snippet: Antibody , Goat F(ab')2 Anti-Mouse IgG2b, Human ads-FITC (Goat Polyclonal) , Southern Biotech , Cat# 1092-02 , Flow cytometry (1/300).

Techniques: Concentration Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Expressing, Flow Cytometry

Journal: eLife

Article Title: Protein kinase Cδ is essential for the IgG response against T-cell-independent type 2 antigens and commensal bacteria

doi: 10.7554/eLife.72116

Figure Lengend Snippet: ( A ) Immunoblot analysis of the indicated molecules in Igk −/− B1-8 flox/+ B cells stimulated with NP-CGG or NP-Ficoll for the indicated time periods. ( B–D ) Prkcd + /+ Igk −/− B1-8 flox/+ or Prkcd −/− Igk −/− B1-8 flox/+ B cells were stimulated with NP-Ficoll and IL-1α, IL-1β, or IFNα. The B cells were labeled with CellTrace Violet (CTV) before culture in C. ( B ) Enzyme-linked immunosorbent assay (ELISA) of IgM and IgG3 in the culture supernatants on day 5. AU, arbitrary units. ( C ) Representative flow cytometric plots of the B cells on day 3 showing percentages of IgG3 + B cells (left). The frequencies of IgG3 + cells at each cell division number (right). ( D ) qRT-PCR analysis of the circle Iγ3-Cµ and Aicda transcripts on day 2. Data are means ± standard deviations (SDs) of two ( B ) or three ( C ) biological replicates or three technical replicates ( D ). The data are representative of at least three ( A and B ) or two ( C and D ) independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001; p values were calculated by unpaired multiple t -test ( B and C ). Figure 2—source data 1. Source data for . Figure 2—source data 2. Source data for . Figure 2—source data 3. Source data for . Figure 2—source data 4. Source data for . Figure 2—source data 5. Source data for .

Article Snippet: Antibody , Goat F(ab')2 Anti-Mouse IgG2b, Human ads-FITC (Goat Polyclonal) , Southern Biotech , Cat# 1092-02 , Flow cytometry (1/300).

Techniques: Western Blot, Labeling, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

Journal: eLife

Article Title: Protein kinase Cδ is essential for the IgG response against T-cell-independent type 2 antigens and commensal bacteria

doi: 10.7554/eLife.72116

Figure Lengend Snippet: ( A and B ) Prkcd + /+ Igk −/− B1-8 flox/+ or Prkcd −/− Igk −/− B1-8 flox/+ naive B cells were cultured with medium alone (–) or NP-Ficoll. ( A ) 3 H-thymidine incorporation on day 3. ( B ) IgM concentration in the culture supernatant on day 4. ( C ) The histogram of CellTrace Violet (CTV) in cells analyzed in . ( D–G ) Prkcd + /+ Igk −/− B1-8 flox/+ or Prkcd − /− Igk −/− B1-8 flox/+ naive B cells were cultured with NP-Ficoll and LPS, R-848, or CpG ( D and E ) or LPS, R-848, or CpG alone ( F and G ). ( D and F ) IgM and IgG concentrations in the culture supernatants on day 3. ( E and G ) qRT-PCR analysis of Aicda transcripts on day 2. Data are means ± standard deviation (SD) of three ( A ) or two to three ( B ), and two ( D and F ) biological replicates, or three technical replicates ( E and G ). The data are representative of two independent experiments. *p < 0.05; ***p < 0.001; p values were calculated by unpaired multiple t -test ( A, B, and D ). Figure 2—figure supplement 1—source data 1. Source data for .

Article Snippet: Antibody , Goat F(ab')2 Anti-Mouse IgG2b, Human ads-FITC (Goat Polyclonal) , Southern Biotech , Cat# 1092-02 , Flow cytometry (1/300).

Techniques: Cell Culture, Concentration Assay, Quantitative RT-PCR, Standard Deviation

Journal: eLife

Article Title: Protein kinase Cδ is essential for the IgG response against T-cell-independent type 2 antigens and commensal bacteria

doi: 10.7554/eLife.72116

Figure Lengend Snippet: ( A ) Flow cytometric analysis of spleen cells and peritoneal cavity cells of Cd19 cre/+ Prkcd +/+ and Cd19 cre/+ Prkcd fl/fl mice. Gating strategy of follicular B cells (Fo B; CD19 + CD43 − CD21 + CD23 + ), marginal zone B cells (MZ B; CD19 + CD43 − CD21 high CD23 low ), B1a cells (CD19 + CD43 + CD5 + ), and B1b cells (CD19 + CD43 + CD5 − ) in the spleen is shown (left). The numbers of Fo B, MZ B, B1a, and B1b cells in spleen (middle) and peritoneal cavity (right) are plotted. ( B ) Enzyme-linked immunosorbent assay (ELISA) of anti-NP IgM, IgG1, and IgG3 in the sera of Cd19 cre/+ Prkcd +/+ and Cd19 cre/+ Prkcd fl/fl mice at indicated weeks after immunization with NP-CGG in alum. AU, arbitrary units. Small horizontal bars are the means of four ( A ) or six ( B ) biological replicates. Each symbol represents an individual mouse. The data are representative of three ( A ) or two ( B ) independent experiments. *p < 0.05; ****p < 0.0001; p values were calculated by unpaired multiple t- test. Figure 3—figure supplement 1—source data 1. Source data for .

Article Snippet: Antibody , Goat F(ab')2 Anti-Mouse IgG2b, Human ads-FITC (Goat Polyclonal) , Southern Biotech , Cat# 1092-02 , Flow cytometry (1/300).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: eLife

Article Title: Protein kinase Cδ is essential for the IgG response against T-cell-independent type 2 antigens and commensal bacteria

doi: 10.7554/eLife.72116

Figure Lengend Snippet: ( A–C ) Cd19 cre/+ Prkcd +/+ and Cd19 cre/+ Prkcd f/f mice were immunized with NP-Ficoll. ( A ) Enzyme-linked immunosorbent assay (ELISA) of serum anti-NP IgM and IgG3 at the indicated weeks. ( B ) ELISA of serum anti-NP IgG1, IgG2b, and IgG2c at 2 weeks after immunization. ( C ) Representative flow cytometric plots of the spleen cells on day 7 after immunization. The numbers indicate the percentage of cells in each gate (left). The frequencies of IgM + , IgG2b + , and IgG3 + cells among NP-binding plasma cells (PCs; NP + CD138 + ; middle) and the numbers of such cells per 1 × 10 7 total lymphocytes (right) are plotted. ( D ) ELISA of anti-PPS3 IgM and IgG3 in the serum of Cd19 cre/+ Prkcd +/+ and Cd19 cre/+ Prkcd f/f mice at the indicated weeks after immunization with PPS3. Results are presented in AU, arbitrary units ( A, B, and D ). Small horizontal bars are the means of six to eight (A and B), five ( C ), and eight ( D ) biological replicates. Each symbol represents an individual mouse. The data are representative of three ( A and C ) or two ( B and D ) independent experiments. *p < 0.05; **p < 0.01; ****p < 0.0001; p values were calculated by unpaired multiple ( A, C, and D ) or two-tailed unpaired Welch’s t -test ( B ). Figure 3—source data 1. Source data for .

Article Snippet: Antibody , Goat F(ab')2 Anti-Mouse IgG2b, Human ads-FITC (Goat Polyclonal) , Southern Biotech , Cat# 1092-02 , Flow cytometry (1/300).

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Clinical Proteomics, Two Tailed Test

Journal: eLife

Article Title: Protein kinase Cδ is essential for the IgG response against T-cell-independent type 2 antigens and commensal bacteria

doi: 10.7554/eLife.72116

Figure Lengend Snippet: ( A and B ) B cells purified from Prkcd +/+ Cd19 cre/+ B1-8 hi or Prkcd f/f Cd19 cre/+ B1-8 hi mice were labeled with CellTrace Violet (CTV) and transferred into B6 mice, which were immunized with NP-Ficoll on the next day. Spleen cells were analyzed 3 days later. ( A ) Representative flow cytometric plots of the spleen cells with the numbers indicating percentages of the cells within the neighboring gates (left). The frequency of IgG3 + cells among whole donor B cells (CD45.1 + CD19 + CD138 – ) (middle) and at each cell division number (right) are shown. ( B ) qRT-PCR analysis of the indicated transcripts in the donor B cells collected as in . ( C ) Prkcd +/+ Cd19 cre/+ B1-8 hi or Prkcd f/f Cd19 cre/+ B1-8 hi B cells transduced with an empty vector (Ev) or vectors expressing AID or PKCδ were transferred into B6 mice that had been immunized with NP-Ficoll on the previous day as in . Representative flow cytometry plots of the donor cells transduced with the vectors (CD45.1 + GFP + , gated as in ) (left) and the frequency of IgG3 + cells among the CD45.1 + GFP + cells (right) on day 3 after transfer. Small horizontal bars are the means of five ( A ) or six to nine ( C ) biological replicates. Each symbol represents an individual mouse (A, middle; C ). The symbols are the means of five biological replicates (A, right). Data are means ± standard deviations (SDs) of three technical replicates pooled from five mice ( B ). The data are representative of three ( A ) or two ( B ) independent experiments or is pooled from two independent experiments ( C ). ns, not significant (p > 0.05); **p < 0.01; ****p < 0.0001; p values were calculated by unpaired multiple t -test ( A ) or one-way analysis of variance (ANOVA) with Tukey’s test ( C ). Figure 4—source data 1. Source data for .

Article Snippet: Antibody , Goat F(ab')2 Anti-Mouse IgG2b, Human ads-FITC (Goat Polyclonal) , Southern Biotech , Cat# 1092-02 , Flow cytometry (1/300).

Techniques: Purification, Labeling, Quantitative RT-PCR, Transduction, Plasmid Preparation, Expressing, Flow Cytometry

Journal: eLife

Article Title: Protein kinase Cδ is essential for the IgG response against T-cell-independent type 2 antigens and commensal bacteria

doi: 10.7554/eLife.72116

Figure Lengend Snippet: ( A ) Sorting strategy for donor B cells used in . The starting population was spleen cells of B6 mice transferred with Prkcd +/+ Cd19 cre/+ B1-8 hi or Prkcd f/f Cd19 cre/+ B1-8 hi , CD45.1 + B cells and immunized with NP-Ficoll 3 days previously. CD45.1 + cells were enriched by a MACS system (left) and then donor B cells (CD45.1 + CD19 + CD138 − ) were further sorted by flow cytometry (right). ( B and C ) Strategy for retrovirus (Rv) transduction and the analysis of the TI-2 response (B, left). B cells collected from Prkcd +/+ Cd19 cre/+ B1-8 hi or Prkcd f/f Cd19 cre/+ B1-8 hi mice immunized with NP-Ficoll 1 day previously were infected with Rv and cultured for 1 day. Some Rv-transduced cells were further cultured in medium for 6 hr and analyzed by flow cytometry. Representative data of the cells transduced with pMXs-IRES-GFP empty vector (Ev) is shown (B, right). Other Rv-transduced cells were transferred into B6 mice that had been immunized with NP-Ficoll on the previous day. Spleen cells of recipient mice were analyzed or used for cell sorting 3 or 4 days later. Gating strategy of donor cells transduced with vectors (CD45.1 + GFP + ) and the expression profile of IgG3 are shown (C, left). After the enrichment of CD45.1 + cells by a MACS system, the vector-transduced donor B cells (CD45.1 + GFP + CD19 + CD138 – ) were sorted as shown (C, right). The data are representative of at least three independent experiments.

Article Snippet: Antibody , Goat F(ab')2 Anti-Mouse IgG2b, Human ads-FITC (Goat Polyclonal) , Southern Biotech , Cat# 1092-02 , Flow cytometry (1/300).

Techniques: Flow Cytometry, Transduction, Infection, Cell Culture, Plasmid Preparation, FACS, Expressing

Journal: eLife

Article Title: Protein kinase Cδ is essential for the IgG response against T-cell-independent type 2 antigens and commensal bacteria

doi: 10.7554/eLife.72116

Figure Lengend Snippet: ( A ) qRT-PCR analysis of the transcripts of the indicated genes in Prkcd +/+ Cd19 cre/+ B1-8 hi or Prkcd f/f Cd19 cre/+ B1-8 hi donor B cells (CD45.1 + CD19 + CD138 − ) purified as in from the recipient mice immunized with NP-Ficoll 3 days previously. Shown is the relative expression of each gene in Prkcd f/f Cd19 cre/+ cells to that in Prkcd +/+ Cd19 cre/+ cells. ( B ) qRT-PCR analysis of Batf transcripts in Prkcd + /+ Igk −/− B1-8 flox/+ or Prkcd −/− Igk −/− B1-8 flox/+ B cells cultured with medium alone (–) or with the indicated stimuli for 2 days. ( C ) Immunoblot analysis in Prkcd + /+ Igk −/− B1-8 flox/+ or Prkcd −/− Igk −/− B1-8 flox/+ B cells stimulated with NP-Ficoll for the indicated times. ( D and E ) B1-8 hi B cells transduced with knockdown vectors for luciferase (shControl) or BATF (shBATF) were transferred into B6 mice that had been immunized with NP-Ficoll on the previous day as in , and their spleen cells were analyzed on day 4 after transfer. ( D ) qRT-PCR analysis of Aicda and Batf transcripts in the vector-transduced donor B cells (CD45.1 + GFP + CD19 + CD138 − ) collected as in . ( E ) Representative flow cytometric plots of the transduced donor cells with the numbers indicating the percentage of IgG3 + cells (left) and the frequency of the IgG3 + cells among such cells (right) gated as in . ( F and G ) Prkcd f/f Cd19 cre/+ B1-8 hi B cells transduced with Ev or vectors expressing BATF or PKCδ were transferred into B6 mice that had been immunized with NP-Ficoll on the previous day, and their spleen cells were analyzed 3 days after transfer. ( F ) qRT-PCR analysis of Aicda transcripts in the vector-transduced donor B cells. ( G ) Representative flow cytometric plots of the transduced donor cells with the numbers indicating the percentage of IgG3 + cells (left) and the frequency of the IgG3 + cells among such cells (right). Data are means ± standard deviations (SDs) of three technical replicates ( A, B, D, and F ). Samples were pooled from five to eight mice ( D and F ). Small horizontal bars are the means of four ( E ) or eight to nine ( G ) biological replicates. Each symbol represents an individual mouse ( E and G ). The data are representative of two independent experiments ( A–F ) or are pooled from two independent experiments ( G ). ***p < 0.001; ****p < 0.0001; p values were calculated by two-tailed unpaired Student’s t -test ( E ) or one-way analysis of variance (ANOVA) with Tukey’s test ( G ). Figure 5—source data 1. Source data for . Figure 5—source data 2. Source data for . Figure 5—source data 3. Source data for . Figure 5—source data 4. Source data for . Figure 5—source data 5. Source data for .

Article Snippet: Antibody , Goat F(ab')2 Anti-Mouse IgG2b, Human ads-FITC (Goat Polyclonal) , Southern Biotech , Cat# 1092-02 , Flow cytometry (1/300).

Techniques: Quantitative RT-PCR, Purification, Expressing, Cell Culture, Western Blot, Transduction, Knockdown, Luciferase, Plasmid Preparation, Two Tailed Test

Journal: eLife

Article Title: Protein kinase Cδ is essential for the IgG response against T-cell-independent type 2 antigens and commensal bacteria

doi: 10.7554/eLife.72116

Figure Lengend Snippet: Cd19 cre/+ Prkcd +/+ and Cd19 cre/+ Prkcd f/f mice were cohoused for at least 4 weeks and treated with 3 % dextran sodium sulfate (DSS) for 7 days. ( A ) Serum IgM, IgG1, IgG2b, and IgG3 against fecal bacteria after cohousing were quantified by enzyme-linked immunosorbent assay (ELISA). AU, arbitrary units. ( B ) Colony-forming unit (CFU) of aerobes and anaerobes in the blood 7 days after DSS treatment. ( C ) Kaplan–Meier survival plot of 16 ( Cd19 cre/+ Prkcd +/+ ) and 12 ( Cd19 cre/+ Prkcdf f/f ) mice at indicated days. Small horizontal bars are the means of 7 ( A ), or 15 ( Cd19 cre/+ Prkcd +/+ ) or 11 ( Cd19 cre/+ Prkcdf f/f ) in ( B ), biological replicates. Data were obtained from 16 ( Cd19 cre/+ Prkcd +/+ ) or 12 ( Cd19 cre/+ Prkcdf f/f ) mice in ( C ). Each symbol represents an individual mouse ( A and B ). The data are representative of two independent experiments ( A ) or pooled from two independent experiments ( B and C ). **p < 0.01; ***p < 0.001; p values were calculated by two-tailed unpaired Welch’s t -test ( A ), unpaired multiple t -test ( B ), or log-rank test ( C ). Figure 6—source data 1. Source data for .

Article Snippet: Antibody , Goat F(ab')2 Anti-Mouse IgG2b, Human ads-FITC (Goat Polyclonal) , Southern Biotech , Cat# 1092-02 , Flow cytometry (1/300).

Techniques: Bacteria, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: eLife

Article Title: Protein kinase Cδ is essential for the IgG response against T-cell-independent type 2 antigens and commensal bacteria

doi: 10.7554/eLife.72116

Figure Lengend Snippet: B-cell receptor (BCR) stimulation with a TI-2 antigen (Ag) activates PKCδ signaling and induces the expression of BATF, which works cooperatively with secondary signals induced by IL-1α/β, IFNα, or TLR ligands and drives the transcription of activation-induced cytidine deaminase (AID) to induce IgG CSR.

Article Snippet: Antibody , Goat F(ab')2 Anti-Mouse IgG2b, Human ads-FITC (Goat Polyclonal) , Southern Biotech , Cat# 1092-02 , Flow cytometry (1/300).

Techniques: Expressing, Activation Assay