Structured Review

The Jackson Laboratory wt c57bl 6
Survival curve of WT C3H and TLR4-deficient mice (A) or WT <t>C57BL/6</t> and TNF-α −/− mice (B) inoculated with the indicated CFU of B. bronchiseptica. Symbols: ⋄, WT; ♦, TLR4 deficient and TNF-α −/− . n = 8 to 10 per group.
Wt C57bl 6, supplied by The Jackson Laboratory, used in various techniques. Bioz Stars score: 91/100, based on 107 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/wt c57bl 6/product/The Jackson Laboratory
Average 91 stars, based on 107 article reviews
Price from $9.99 to $1999.99
wt c57bl 6 - by Bioz Stars, 2021-01
91/100 stars

Images

1) Product Images from "Toll-Like Receptor 4-Dependent Early Elicited Tumor Necrosis Factor Alpha Expression Is Critical for Innate Host Defense against Bordetella bronchiseptica"

Article Title: Toll-Like Receptor 4-Dependent Early Elicited Tumor Necrosis Factor Alpha Expression Is Critical for Innate Host Defense against Bordetella bronchiseptica

Journal: Infection and Immunity

doi: 10.1128/IAI.72.11.6650-6658.2004

Survival curve of WT C3H and TLR4-deficient mice (A) or WT C57BL/6 and TNF-α −/− mice (B) inoculated with the indicated CFU of B. bronchiseptica. Symbols: ⋄, WT; ♦, TLR4 deficient and TNF-α −/− . n = 8 to 10 per group.
Figure Legend Snippet: Survival curve of WT C3H and TLR4-deficient mice (A) or WT C57BL/6 and TNF-α −/− mice (B) inoculated with the indicated CFU of B. bronchiseptica. Symbols: ⋄, WT; ♦, TLR4 deficient and TNF-α −/− . n = 8 to 10 per group.

Techniques Used: Mouse Assay

2) Product Images from "Blockade of self-reactive IgM significantly reduces injury in a murine model of acute myocardial infarction"

Article Title: Blockade of self-reactive IgM significantly reduces injury in a murine model of acute myocardial infarction

Journal: Cardiovascular Research

doi: 10.1093/cvr/cvq141

Effect of N2 treatment on collagen deposition in C57BL/6 mice. ( A ) C57BL/6 mice were injected iv with NS or N2 peptide and subjected to 1 h ischaemia and 5-day reperfusion. Hearts were collected after 5-day reperfusion and cryosections were stained with
Figure Legend Snippet: Effect of N2 treatment on collagen deposition in C57BL/6 mice. ( A ) C57BL/6 mice were injected iv with NS or N2 peptide and subjected to 1 h ischaemia and 5-day reperfusion. Hearts were collected after 5-day reperfusion and cryosections were stained with

Techniques Used: Mouse Assay, Injection, Staining

( A ) Quantitation of IgM staining in the area of risk (AR) and non-risk (NR) area of NS-, N2-, and 21G6 F(ab′) 2 -treated C57BL/6 mice. ( B ) Effect of 21G6 F(ab′) 2 on cTnI levels following I/R. 21G6 F(ab′) 2 (150 µg) was injected
Figure Legend Snippet: ( A ) Quantitation of IgM staining in the area of risk (AR) and non-risk (NR) area of NS-, N2-, and 21G6 F(ab′) 2 -treated C57BL/6 mice. ( B ) Effect of 21G6 F(ab′) 2 on cTnI levels following I/R. 21G6 F(ab′) 2 (150 µg) was injected

Techniques Used: Quantitation Assay, Staining, Mouse Assay, Injection

Effect of N2 peptide and 21G6 F(ab′) 2 on myocardial I/R injury. Representative cryosections of mock-, NS-, N2-, or 21G6 F(ab′) 2 -treated C57BL/6 mice subjected to I/R. Cryosections were stained with H E, anti-C3d-FITC, and anti-IgM-568.
Figure Legend Snippet: Effect of N2 peptide and 21G6 F(ab′) 2 on myocardial I/R injury. Representative cryosections of mock-, NS-, N2-, or 21G6 F(ab′) 2 -treated C57BL/6 mice subjected to I/R. Cryosections were stained with H E, anti-C3d-FITC, and anti-IgM-568.

Techniques Used: Mouse Assay, Staining

Effect of N2 treatment on neutrophil and monocyte infiltration of injured myocardium. C57BL/6 mice were injected with NS or N2 peptide and subjected to I/R. Inflammatory cell populations were analysed by FACS of mock-, NS-, or N2-treated hearts. ( A ) Representative
Figure Legend Snippet: Effect of N2 treatment on neutrophil and monocyte infiltration of injured myocardium. C57BL/6 mice were injected with NS or N2 peptide and subjected to I/R. Inflammatory cell populations were analysed by FACS of mock-, NS-, or N2-treated hearts. ( A ) Representative

Techniques Used: Mouse Assay, Injection, FACS

3) Product Images from "Autocrine Effects of Tumor-Derived Complement"

Article Title: Autocrine Effects of Tumor-Derived Complement

Journal: Cell reports

doi: 10.1016/j.celrep.2014.02.014

Cancer Cells Originated from Different Organs Secrete Complement Proteins that Enhance Cell Proliferation (A) Quantification of C3 mRNA in h breast, ovarian, lung, and endometrium cancer cell lines using quantitative real-time PCR (n = 3). (B and C) Reducing C3 gene expression in (B) H226 human squamous cell lung cancer and in (C) Hec265 human endometrium cancer cell lines using C3 siRNA reduced proliferation, migration, and invasion of these cells in vitro. Results of three independent experiments (each of them in triplicate) are summarized as bar graphs (*p ≤ 0.01 and **p ≤ 0.001, t test). (D) The relative abundance of AKT mRNA in total mRNA isolated from SKOV3ip1 cells after 24 hr exposure to C3aR and C5aR agonists was quantified by quantitative real-time PCR, and the result of three experiments are summarized as bar graphs (*p ≤ 0.001). (E) A representative immunoblot on total cell lysate prepared from SKOV3ip1 cells after 48 hr exposure to C3aR and C5aR agonists and inhibitors using antibodies to p-85, AKT, and their phosphorylated forms (n = 3). (F) Immunostaining of tumors induced by ID8-VEGF cells expressing scrambled shRNA or mC3 shRNA in C57BL/6 mice using anti-pAKT antibody. (G) To investigate the effect of AKT or PI3K silencing on C3aR and C5aR agonist-induced enhancement of SKOV3ip1 proliferation, expression of AKT or PI3K in SKOV3ip1 was reduced using AKT or PI3K siRNA (data not shown), and cell proliferation was quantified 48 hr after exposure to C3aR-AG and C5aR-AG and was compared to SKOV3ip1 cells transfected with scrambled siRNA (n = 3; *p ≤ 0.001). (H) C3 mRNA level in the tumor specimens of 75 patients diagnosed with ovarian cancer in MDACC was determined using quantitative real-time PCR and correlated with their OS (p = 0.004) and presented as Kaplan-Meier survival curve. (I) Correlation between expression of C5aR in tumor and OS in 562 patients with ovarian cancer in TCGA database (p = 0.019). (J) The amount of C5aR mRNA in tumors resected from 167 patients with a diagnosis of lung squamous cell carcinoma was quantified using quantitative real-time PCR and was correlated to the OS of these patients as documented in TCGA database. The results are shown as Kaplan-Meier survival curve (p = 0.026).
Figure Legend Snippet: Cancer Cells Originated from Different Organs Secrete Complement Proteins that Enhance Cell Proliferation (A) Quantification of C3 mRNA in h breast, ovarian, lung, and endometrium cancer cell lines using quantitative real-time PCR (n = 3). (B and C) Reducing C3 gene expression in (B) H226 human squamous cell lung cancer and in (C) Hec265 human endometrium cancer cell lines using C3 siRNA reduced proliferation, migration, and invasion of these cells in vitro. Results of three independent experiments (each of them in triplicate) are summarized as bar graphs (*p ≤ 0.01 and **p ≤ 0.001, t test). (D) The relative abundance of AKT mRNA in total mRNA isolated from SKOV3ip1 cells after 24 hr exposure to C3aR and C5aR agonists was quantified by quantitative real-time PCR, and the result of three experiments are summarized as bar graphs (*p ≤ 0.001). (E) A representative immunoblot on total cell lysate prepared from SKOV3ip1 cells after 48 hr exposure to C3aR and C5aR agonists and inhibitors using antibodies to p-85, AKT, and their phosphorylated forms (n = 3). (F) Immunostaining of tumors induced by ID8-VEGF cells expressing scrambled shRNA or mC3 shRNA in C57BL/6 mice using anti-pAKT antibody. (G) To investigate the effect of AKT or PI3K silencing on C3aR and C5aR agonist-induced enhancement of SKOV3ip1 proliferation, expression of AKT or PI3K in SKOV3ip1 was reduced using AKT or PI3K siRNA (data not shown), and cell proliferation was quantified 48 hr after exposure to C3aR-AG and C5aR-AG and was compared to SKOV3ip1 cells transfected with scrambled siRNA (n = 3; *p ≤ 0.001). (H) C3 mRNA level in the tumor specimens of 75 patients diagnosed with ovarian cancer in MDACC was determined using quantitative real-time PCR and correlated with their OS (p = 0.004) and presented as Kaplan-Meier survival curve. (I) Correlation between expression of C5aR in tumor and OS in 562 patients with ovarian cancer in TCGA database (p = 0.019). (J) The amount of C5aR mRNA in tumors resected from 167 patients with a diagnosis of lung squamous cell carcinoma was quantified using quantitative real-time PCR and was correlated to the OS of these patients as documented in TCGA database. The results are shown as Kaplan-Meier survival curve (p = 0.026).

Techniques Used: Real-time Polymerase Chain Reaction, Expressing, Migration, In Vitro, Isolation, Immunostaining, shRNA, Mouse Assay, Transfection

Role of Immunomodulatory Effect of C3 Knockdown on Tumor Growth (A) ID8-VEGF murine ovarian cancer cells stably expressing murine (m) C3 shRNA or control scrambled shRNA were injected into immune-competent C57BL/6 mice (WT and C3 −/− ). Cancer cells with C3 knockdown generated smaller tumors in both WT and C3 −/− mice. **p ≤ 0.0001. (B) Representative immunostaining for CD8 and CD11b in tumors induced by mC3 shRNA-expressing or scrambled shRNA-expressing ID8-VEGF cells in WT mice. Scale bar, 100 μm. (C) The number of cytotoxic T cells (CD8+) and myeloid cells (CD11b+) were determined by counting cells in three high-power fields per tumor nodule per mouse and in five mice per group, and the results are summarized as bar graphs (28 CD8+ cells/HPF in scrambled shRNA-expressing and 358 CD8+/HPF in C3 shRNA-expressing cell-induced tumors; 82 CD11b+ cells/HPF in scrambled shRNA-expressing and average number of 25 CD11b+ cells/HPF in C3 shRNA-expressing cell-induced tumors, n = 15 HPF; *p = 0.001, t test). (D) ID8-VEGF cells stably expressing mC3 shRNA or control scrambled shRNA were injected to CD8 −/− and WT mice. There was no significant difference in tumor size after injection with scrambled shRNA-expressing cancer cells between CD8 −/− and WT mice (0.60 ± 0.23 g versus 0.77 ± 0.17 g, respectively; n = 10 mice/ group; p = 0.19). C3 silencing by mC3 shRNA in cancer cells resulted in 98% reduction in tumor weight in both WT and CD8 −/− mice (*p ≤ 0.001). (E) A representative necropsy in mice showing the presence of tumor nodules on viscera and peritoneal surfaces (surrounded by dashed lines) in WT and CD8 −/− mice injected with ID8-VEGF cells expressing scrambled shRNA and lack of tumor nodules in those injected with mC3 shRNA-expressing cancer cells. Arrows show tumor nodules on the peritoneal surface of diaphragm.
Figure Legend Snippet: Role of Immunomodulatory Effect of C3 Knockdown on Tumor Growth (A) ID8-VEGF murine ovarian cancer cells stably expressing murine (m) C3 shRNA or control scrambled shRNA were injected into immune-competent C57BL/6 mice (WT and C3 −/− ). Cancer cells with C3 knockdown generated smaller tumors in both WT and C3 −/− mice. **p ≤ 0.0001. (B) Representative immunostaining for CD8 and CD11b in tumors induced by mC3 shRNA-expressing or scrambled shRNA-expressing ID8-VEGF cells in WT mice. Scale bar, 100 μm. (C) The number of cytotoxic T cells (CD8+) and myeloid cells (CD11b+) were determined by counting cells in three high-power fields per tumor nodule per mouse and in five mice per group, and the results are summarized as bar graphs (28 CD8+ cells/HPF in scrambled shRNA-expressing and 358 CD8+/HPF in C3 shRNA-expressing cell-induced tumors; 82 CD11b+ cells/HPF in scrambled shRNA-expressing and average number of 25 CD11b+ cells/HPF in C3 shRNA-expressing cell-induced tumors, n = 15 HPF; *p = 0.001, t test). (D) ID8-VEGF cells stably expressing mC3 shRNA or control scrambled shRNA were injected to CD8 −/− and WT mice. There was no significant difference in tumor size after injection with scrambled shRNA-expressing cancer cells between CD8 −/− and WT mice (0.60 ± 0.23 g versus 0.77 ± 0.17 g, respectively; n = 10 mice/ group; p = 0.19). C3 silencing by mC3 shRNA in cancer cells resulted in 98% reduction in tumor weight in both WT and CD8 −/− mice (*p ≤ 0.001). (E) A representative necropsy in mice showing the presence of tumor nodules on viscera and peritoneal surfaces (surrounded by dashed lines) in WT and CD8 −/− mice injected with ID8-VEGF cells expressing scrambled shRNA and lack of tumor nodules in those injected with mC3 shRNA-expressing cancer cells. Arrows show tumor nodules on the peritoneal surface of diaphragm.

Techniques Used: Stable Transfection, Expressing, shRNA, Injection, Mouse Assay, Generated, Immunostaining

Ovarian Cancer Cells Secrete Complement Proteins, which Enhance Tumor Growth (A) We measured total tumor weight in an orthotopic murine model of ovarian cancer induced by ID8-VEGF murine ovarian cancer cells in C3 −/− and WT control mice, both in C57BL/6 background. n.s, not significant. (B) Immunostaining of tumors induced by ID8-VEGF in WT and C3 −/− mice, using anti-C3 antibody compared to negative control stain (secondary antibody alone). Scale bar length is 100 μm. (C) Quantitative real-time PCR for C3 mRNA on RNA isolated from murine and human ovarian cancer cell lines. Expression of C3 mRNA in cancer cell lines was compared to that in MOECs and normal human ovarian surface epithelial cell lines (HIO 180) (n = 3; **p ≤ 0.01, t test). (D) C3 gene knockdown in SKOV3ip1 human ovarian cancer cells using C3 siRNA, reduced proliferation, migration, and invasion of these cells in vitro. Results of three independent experiments (each of them in triplicate) are summarized as bar graphs (**p ≤ 0.01, t test). (E) C3 gene knockdown in SKOV3ip1-induced tumors by intraperitoneal injection of hC3 siRNA into tumor-bearing NU/NU mice reduced total weight (*p = 0.017) and number of tumor nodules (*p = 0.05). (F) Representative immunostaining for C3, Ki67, and CD31 in tumors resected from hC3 siRNA-injected and scrambled siRNA-injected mice. Scale bar, 100 μm. (G) The proliferation index in resected tumors was quantified as the percentage of Ki67 positivity shown in dot plots (39% in C3 siRNA versus 74% in scrambled siRNA, n = 5 mice in each group; *p = 0.05, t test). The number of blood vessels in resected tumors was quantified by counting the number of CD31+ lumen structures in five high-power fields (HPFs) per section and in five sections per tumor nodule and in five mice per group. Average number of CD31+ lumens per HPF is shown as dot plots (22/HPF in C3 siRNA versus 42/HPF in scrambled siRNA; *p = 0.05, t test). (H) We investigated the effect of complement on proliferation of endothelial cells by measuring the proliferation rate of RF24 endothelial cells after transfection with C3 siRNA. C3 knockdown did not reduce the proliferation rate in RF24 endothelial cells (n = 3; p = 0.07, t test).
Figure Legend Snippet: Ovarian Cancer Cells Secrete Complement Proteins, which Enhance Tumor Growth (A) We measured total tumor weight in an orthotopic murine model of ovarian cancer induced by ID8-VEGF murine ovarian cancer cells in C3 −/− and WT control mice, both in C57BL/6 background. n.s, not significant. (B) Immunostaining of tumors induced by ID8-VEGF in WT and C3 −/− mice, using anti-C3 antibody compared to negative control stain (secondary antibody alone). Scale bar length is 100 μm. (C) Quantitative real-time PCR for C3 mRNA on RNA isolated from murine and human ovarian cancer cell lines. Expression of C3 mRNA in cancer cell lines was compared to that in MOECs and normal human ovarian surface epithelial cell lines (HIO 180) (n = 3; **p ≤ 0.01, t test). (D) C3 gene knockdown in SKOV3ip1 human ovarian cancer cells using C3 siRNA, reduced proliferation, migration, and invasion of these cells in vitro. Results of three independent experiments (each of them in triplicate) are summarized as bar graphs (**p ≤ 0.01, t test). (E) C3 gene knockdown in SKOV3ip1-induced tumors by intraperitoneal injection of hC3 siRNA into tumor-bearing NU/NU mice reduced total weight (*p = 0.017) and number of tumor nodules (*p = 0.05). (F) Representative immunostaining for C3, Ki67, and CD31 in tumors resected from hC3 siRNA-injected and scrambled siRNA-injected mice. Scale bar, 100 μm. (G) The proliferation index in resected tumors was quantified as the percentage of Ki67 positivity shown in dot plots (39% in C3 siRNA versus 74% in scrambled siRNA, n = 5 mice in each group; *p = 0.05, t test). The number of blood vessels in resected tumors was quantified by counting the number of CD31+ lumen structures in five high-power fields (HPFs) per section and in five sections per tumor nodule and in five mice per group. Average number of CD31+ lumens per HPF is shown as dot plots (22/HPF in C3 siRNA versus 42/HPF in scrambled siRNA; *p = 0.05, t test). (H) We investigated the effect of complement on proliferation of endothelial cells by measuring the proliferation rate of RF24 endothelial cells after transfection with C3 siRNA. C3 knockdown did not reduce the proliferation rate in RF24 endothelial cells (n = 3; p = 0.07, t test).

Techniques Used: Mouse Assay, Immunostaining, Negative Control, Staining, Real-time Polymerase Chain Reaction, Isolation, Expressing, Migration, In Vitro, Injection, Transfection

4) Product Images from "CD8? Dendritic Cells Drive Establishment of HSV-1 Latency"

Article Title: CD8? Dendritic Cells Drive Establishment of HSV-1 Latency

Journal: PLoS ONE

doi: 10.1371/journal.pone.0093444

Quantitation of gB DNA and LAT RNA in trigeminal ganglia of HSV-1 latently-infected mice. Wild-type (WT) C57BL/6, β 2 m −/− , or CD8α −/− mice were ocularly-infected with HSV-1 strain McKrae (LAT(+)). On day 28 PI, TG were harvested from latently infected mice. Quantitative PCR and RT-PCR was performed on each individual mouse TG. In each experiment, an estimated relative copy number of the HSV-1 gB (for viral DNA) and LAT (for viral RNA) were calculated using standard curves generated from pGem-gB1 and pGem5317, respectively. Briefly, DNA template was serially diluted 10-fold such that 5 μl contained from 10 3 to 10 11 copies of gB, then subjected to TaqMan PCR with the same set of primers. By comparing the normalized threshold cycle of each sample to the threshold cycle of the standard, the copy number for each reaction was determined. GAPDH expression was used to normalize the relative expression of viral (gB) DNA and LAT RNA in the TG. Each data point represents the mean ± SEM from 20 TGs for WT and 18 TGs for β 2 m −/− or CD8α −/− mice from two separate experiments. Panels: A) gB DNA; and B) LAT RNA (the Y-scale for LAT in CD8α −/− mice TG is different than the Y-scale for WT and β 2 m −/− mice).
Figure Legend Snippet: Quantitation of gB DNA and LAT RNA in trigeminal ganglia of HSV-1 latently-infected mice. Wild-type (WT) C57BL/6, β 2 m −/− , or CD8α −/− mice were ocularly-infected with HSV-1 strain McKrae (LAT(+)). On day 28 PI, TG were harvested from latently infected mice. Quantitative PCR and RT-PCR was performed on each individual mouse TG. In each experiment, an estimated relative copy number of the HSV-1 gB (for viral DNA) and LAT (for viral RNA) were calculated using standard curves generated from pGem-gB1 and pGem5317, respectively. Briefly, DNA template was serially diluted 10-fold such that 5 μl contained from 10 3 to 10 11 copies of gB, then subjected to TaqMan PCR with the same set of primers. By comparing the normalized threshold cycle of each sample to the threshold cycle of the standard, the copy number for each reaction was determined. GAPDH expression was used to normalize the relative expression of viral (gB) DNA and LAT RNA in the TG. Each data point represents the mean ± SEM from 20 TGs for WT and 18 TGs for β 2 m −/− or CD8α −/− mice from two separate experiments. Panels: A) gB DNA; and B) LAT RNA (the Y-scale for LAT in CD8α −/− mice TG is different than the Y-scale for WT and β 2 m −/− mice).

Techniques Used: Quantitation Assay, Infection, Mouse Assay, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Generated, Polymerase Chain Reaction, Expressing

Quantitation of HSV-1 latency in CD8β −/− infected mice. Wild-type (WT) C57BL/6 and CD8β −/− mice were infected as described in Materials and Methods. Twenty-eight days PI, TGs from infected mice were harvested and quantitative PCR and RT-PCR were performed on each individual mouse TG. Each data point for gB DNA represents the mean ± SEM from 24 TGs. For LAT RNA, each data point represents 22 TGs for CD8β −/− and 18 TGs for WT mice from two separate experiments. Panels: A) gB DNA; and B) LAT RNA.
Figure Legend Snippet: Quantitation of HSV-1 latency in CD8β −/− infected mice. Wild-type (WT) C57BL/6 and CD8β −/− mice were infected as described in Materials and Methods. Twenty-eight days PI, TGs from infected mice were harvested and quantitative PCR and RT-PCR were performed on each individual mouse TG. Each data point for gB DNA represents the mean ± SEM from 24 TGs. For LAT RNA, each data point represents 22 TGs for CD8β −/− and 18 TGs for WT mice from two separate experiments. Panels: A) gB DNA; and B) LAT RNA.

Techniques Used: Quantitation Assay, Infection, Mouse Assay, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction

5) Product Images from "Regulation of murine NK cell exhaustion through the activation of the DNA damage repair pathway"

Article Title: Regulation of murine NK cell exhaustion through the activation of the DNA damage repair pathway

Journal: JCI Insight

doi: 10.1172/jci.insight.127729

Inhibition of the ATM DNA damage repair pathway delays NCE by increasing NK cell function and survival, which improve antitumor responses after NK cell adoptive transfer therapy. Thy1.2 BM-derived cells were cultured in vitro with IL-2 and treated with DMSO or 7.5 μM ATM inhibitor KU on day 4 of culture. Adherent NK cells were collected at different time points of culture. ( A ) Hierarchical clustering by Euclidean distance analysis of the expression of multiple NK cell markers is shown. ( B ) The NCE phenotype was evaluated on in vitro activated NK cells measured by Eomes MFI and the total percentage of NKG2D and KLRG1 on gated NK cells. ( C ) The percentage of Ki67 is shown. ( D ) BCL2 MFI is shown on gated NK cells. ( E ) The 20S proteasome activity of NK cells is shown. ( F ) The percentage of IFN-γ production after NK1.1 stimulation is shown. ( G ) The percentage of tumor lysis of NK cells against A20 is shown. ( H and I ) C57BL/6 mice received total body radiation (TBI) at the time of tumor i.v. infusion ( H : A20) or 7 days after tumor administration ( I : B16F10), followed by allogeneic ( H ) or syngeneic ( I ) hematopoietic stem cell transplantation (HCT), along with freshly isolated unstimulated NK cells (fresh NK) or 5 day ex vivo expanded activated NK cells in the presence of vehicle control (aNK) or KU (KU aNK). When indicated, mice received low doses of IL-2 (5 × 10 4 IU) for 7 days after NK cell administration. Percentage is of in vivo tumor survival of NKG2D-dependent ( H : A20) or -independent ( I : B16F10) tumor-bearing mice after HCT with adoptive transfer of PBS (control) or NK cells. Data represent 2 or 3 independent experiments done in triplicate (in vitro model) or with 5–8 mice per group (in vivo tumor model) (mean ± SEM). One-way ANOVA (in vitro model) or log-rank test (in vivo tumor model) were used to assess significance (* P
Figure Legend Snippet: Inhibition of the ATM DNA damage repair pathway delays NCE by increasing NK cell function and survival, which improve antitumor responses after NK cell adoptive transfer therapy. Thy1.2 BM-derived cells were cultured in vitro with IL-2 and treated with DMSO or 7.5 μM ATM inhibitor KU on day 4 of culture. Adherent NK cells were collected at different time points of culture. ( A ) Hierarchical clustering by Euclidean distance analysis of the expression of multiple NK cell markers is shown. ( B ) The NCE phenotype was evaluated on in vitro activated NK cells measured by Eomes MFI and the total percentage of NKG2D and KLRG1 on gated NK cells. ( C ) The percentage of Ki67 is shown. ( D ) BCL2 MFI is shown on gated NK cells. ( E ) The 20S proteasome activity of NK cells is shown. ( F ) The percentage of IFN-γ production after NK1.1 stimulation is shown. ( G ) The percentage of tumor lysis of NK cells against A20 is shown. ( H and I ) C57BL/6 mice received total body radiation (TBI) at the time of tumor i.v. infusion ( H : A20) or 7 days after tumor administration ( I : B16F10), followed by allogeneic ( H ) or syngeneic ( I ) hematopoietic stem cell transplantation (HCT), along with freshly isolated unstimulated NK cells (fresh NK) or 5 day ex vivo expanded activated NK cells in the presence of vehicle control (aNK) or KU (KU aNK). When indicated, mice received low doses of IL-2 (5 × 10 4 IU) for 7 days after NK cell administration. Percentage is of in vivo tumor survival of NKG2D-dependent ( H : A20) or -independent ( I : B16F10) tumor-bearing mice after HCT with adoptive transfer of PBS (control) or NK cells. Data represent 2 or 3 independent experiments done in triplicate (in vitro model) or with 5–8 mice per group (in vivo tumor model) (mean ± SEM). One-way ANOVA (in vitro model) or log-rank test (in vivo tumor model) were used to assess significance (* P

Techniques Used: Inhibition, Cell Function Assay, Adoptive Transfer Assay, Derivative Assay, Cell Culture, In Vitro, Expressing, Activity Assay, Lysis, Mouse Assay, Transplantation Assay, Isolation, Ex Vivo, In Vivo

6) Product Images from "Alternatively activated macrophage-derived RELM-? is a negative regulator of type 2 inflammation in the lung"

Article Title: Alternatively activated macrophage-derived RELM-? is a negative regulator of type 2 inflammation in the lung

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20082048

RELM-α is expressed after Sm egg challenge. (A) Lung Retnla expression in naive and Sm egg-challenged C57BL/6 mice. *, P
Figure Legend Snippet: RELM-α is expressed after Sm egg challenge. (A) Lung Retnla expression in naive and Sm egg-challenged C57BL/6 mice. *, P

Techniques Used: Expressing, Mouse Assay

7) Product Images from "IL-1RA regulates immunopathogenesis during fungal-associated allergic airway inflammation"

Article Title: IL-1RA regulates immunopathogenesis during fungal-associated allergic airway inflammation

Journal: JCI Insight

doi: 10.1172/jci.insight.129055

IL-1 receptor antagonist regulates the severity of experimental fungal–associated allergic airway inflammation. C57BL/6 WT and IL-1 receptor antagonist–deficient ( Il1rn –/– ) mice were chronically exposed to A . fumigatus as described in the Methods. Twenty-four hours after the last organism challenge, ( A ) airway (Newtonian) resistance and ( B ) total lung resistance was analyzed via mechanical ventilation using the flexiVent pulmonary function system. The figures illustrate cumulative data from 2 independent studies ( n = 4–5 mice per group per study). Data are expressed as mean ± SEM. *** P
Figure Legend Snippet: IL-1 receptor antagonist regulates the severity of experimental fungal–associated allergic airway inflammation. C57BL/6 WT and IL-1 receptor antagonist–deficient ( Il1rn –/– ) mice were chronically exposed to A . fumigatus as described in the Methods. Twenty-four hours after the last organism challenge, ( A ) airway (Newtonian) resistance and ( B ) total lung resistance was analyzed via mechanical ventilation using the flexiVent pulmonary function system. The figures illustrate cumulative data from 2 independent studies ( n = 4–5 mice per group per study). Data are expressed as mean ± SEM. *** P

Techniques Used: Mouse Assay

In vivo administration of human IL-1RA improves lung function during experimental fungal–associated allergic airway inflammation. C57BL/6 WT mice were chronically exposed to A . fumigatus as described in Methods and treated daily from days 0–16 with 10 mg/kg or 50 mg/kg human recombinant IL-1RA (Kineret/anakinra) or vehicle i.p. Twenty-four hours after the last organism challenge, ( A and C ) Airway (Newtonian) resistance and ( B and D ) total lung resistance was analyzed via mechanical ventilation using the flexiVent pulmonary function system. The figures illustrate cumulative data from 2 independent studies ( n = 4–5 mice per group per study). Data expressed as mean ± SEM. * P
Figure Legend Snippet: In vivo administration of human IL-1RA improves lung function during experimental fungal–associated allergic airway inflammation. C57BL/6 WT mice were chronically exposed to A . fumigatus as described in Methods and treated daily from days 0–16 with 10 mg/kg or 50 mg/kg human recombinant IL-1RA (Kineret/anakinra) or vehicle i.p. Twenty-four hours after the last organism challenge, ( A and C ) Airway (Newtonian) resistance and ( B and D ) total lung resistance was analyzed via mechanical ventilation using the flexiVent pulmonary function system. The figures illustrate cumulative data from 2 independent studies ( n = 4–5 mice per group per study). Data expressed as mean ± SEM. * P

Techniques Used: In Vivo, Mouse Assay, Recombinant

Signaling through the IL-1 receptor worsens lung function during experimental fungal–associated allergic airway inflammation. C57BL/6 and IL-1 receptor–deficient ( Il1r1 –/– ) mice were chronically exposed to A . fumigatus as described in the Methods. Twenty-four hours after the last organism challenge, ( A ) airway (Newtonian) resistance and ( B ) total lung resistance was analyzed via mechanical ventilation using the flexiVent pulmonary function system. The figures illustrate cumulative data from 3 independent studies ( n = 4–5 mice per group per study). Data expressed as mean ± SEM. *** P
Figure Legend Snippet: Signaling through the IL-1 receptor worsens lung function during experimental fungal–associated allergic airway inflammation. C57BL/6 and IL-1 receptor–deficient ( Il1r1 –/– ) mice were chronically exposed to A . fumigatus as described in the Methods. Twenty-four hours after the last organism challenge, ( A ) airway (Newtonian) resistance and ( B ) total lung resistance was analyzed via mechanical ventilation using the flexiVent pulmonary function system. The figures illustrate cumulative data from 3 independent studies ( n = 4–5 mice per group per study). Data expressed as mean ± SEM. *** P

Techniques Used: Mouse Assay

Type 1 responses are differentially regulated by IL-1R1 and IL-1RA during experimental fungal–associated allergic airway inflammation. C57BL/6 WT, IL-1 receptor–deficient ( Il1r1 –/– ), and IL-1 receptor antagonist–deficient ( Il1rn –/– ) mice were chronically exposed to A . fumigatus as in Methods. ( A and B ) Twenty-four hours after last challenge, right lungs were collected and enzymatically digested, and unfractionated lung cells were cocultured with A . fumigatus conidia for 24 hours at a cell/organism ratio of 1:1. IFN-γ, CXCL9, and CXCL10 levels in lung digest cell culture supernatants from ( A ) WT and Il1r1 –/– mice and from ( B ) WT and Il1rn –/– mice were quantified by MilliPlex. The figures illustrate cumulative data from 3 independent studies (3–5 mice per group per time point). For all graphs, quantitative data are represented as a box-and-whisker plot, with bounds ranging from 25th to 75th percentile, the line representing the median, whiskers ranging from minimum to maximum values, and + indicating the mean. * P
Figure Legend Snippet: Type 1 responses are differentially regulated by IL-1R1 and IL-1RA during experimental fungal–associated allergic airway inflammation. C57BL/6 WT, IL-1 receptor–deficient ( Il1r1 –/– ), and IL-1 receptor antagonist–deficient ( Il1rn –/– ) mice were chronically exposed to A . fumigatus as in Methods. ( A and B ) Twenty-four hours after last challenge, right lungs were collected and enzymatically digested, and unfractionated lung cells were cocultured with A . fumigatus conidia for 24 hours at a cell/organism ratio of 1:1. IFN-γ, CXCL9, and CXCL10 levels in lung digest cell culture supernatants from ( A ) WT and Il1r1 –/– mice and from ( B ) WT and Il1rn –/– mice were quantified by MilliPlex. The figures illustrate cumulative data from 3 independent studies (3–5 mice per group per time point). For all graphs, quantitative data are represented as a box-and-whisker plot, with bounds ranging from 25th to 75th percentile, the line representing the median, whiskers ranging from minimum to maximum values, and + indicating the mean. * P

Techniques Used: Mouse Assay, Cell Culture, Whisker Assay

The absence of IL-1R1 and IL-1RA results in varied type 2 responses during experimental fungal–associated allergic airway inflammation. C57BL/6 WT, IL-1 receptor–deficient ( Il1r1 –/– ), and IL-1 receptor antagonist–deficient ( Il1rn –/– ) mice were chronically exposed to A . fumigatus as in Methods. ( A and B ) Twenty-four hours after the last challenge, lung cells from ( A ) WT and Il1r1 –/– mice and ( B ) WT and Il1rn –/– mice were isolated by bronchoalveolar lavage, enumerated, Fc-blocked, stained with a live/dead staining kit, and stained for eosinophils (CD45 + , CD11b + , Siglec F + , Ly-6G – ). The figures illustrate cumulative data from 2–4 independent studies ( n = 4–5 mice per group per study). ( C–F ) Twenty-four after last challenge, right lungs were collected and enzymatically digested, and unfractionated lung cells were cocultured with A . fumigatus conidia for 24 hours at a cell/organism ratio of 1:1. IL-4, IL-5, and CCL11 levels in lung digest cell culture supernatants from ( C and E ) WT and Il1r1 –/– mice and from ( D and F ) WT and Il1rn –/– mice were quantified by MilliPlex. The figures illustrate cumulative data from 3–4 independent studies ( n = 3–5 mice per group per study). ( G–J ) Twenty four hours after last challenge, left lungs were collected and homogenized, and CCL17, CCL22, and IL-33 levels were quantified by ELISA in clarified lung homogenates from ( G and I ) WT and Il1r1 –/– mice and from ( H and J ) WT and Il1rn –/– mice. The figures illustrate cumulative data from 2–3 independent studies ( n = 3–5 mice per group per study). For all graphs, quantitative data are represented as a box-and-whisker plot, with bounds ranging from 25th to 75th percentile, the line representing the median, whiskers ranging from minimum to maximum values, and + indicating the mean. * P
Figure Legend Snippet: The absence of IL-1R1 and IL-1RA results in varied type 2 responses during experimental fungal–associated allergic airway inflammation. C57BL/6 WT, IL-1 receptor–deficient ( Il1r1 –/– ), and IL-1 receptor antagonist–deficient ( Il1rn –/– ) mice were chronically exposed to A . fumigatus as in Methods. ( A and B ) Twenty-four hours after the last challenge, lung cells from ( A ) WT and Il1r1 –/– mice and ( B ) WT and Il1rn –/– mice were isolated by bronchoalveolar lavage, enumerated, Fc-blocked, stained with a live/dead staining kit, and stained for eosinophils (CD45 + , CD11b + , Siglec F + , Ly-6G – ). The figures illustrate cumulative data from 2–4 independent studies ( n = 4–5 mice per group per study). ( C–F ) Twenty-four after last challenge, right lungs were collected and enzymatically digested, and unfractionated lung cells were cocultured with A . fumigatus conidia for 24 hours at a cell/organism ratio of 1:1. IL-4, IL-5, and CCL11 levels in lung digest cell culture supernatants from ( C and E ) WT and Il1r1 –/– mice and from ( D and F ) WT and Il1rn –/– mice were quantified by MilliPlex. The figures illustrate cumulative data from 3–4 independent studies ( n = 3–5 mice per group per study). ( G–J ) Twenty four hours after last challenge, left lungs were collected and homogenized, and CCL17, CCL22, and IL-33 levels were quantified by ELISA in clarified lung homogenates from ( G and I ) WT and Il1r1 –/– mice and from ( H and J ) WT and Il1rn –/– mice. The figures illustrate cumulative data from 2–3 independent studies ( n = 3–5 mice per group per study). For all graphs, quantitative data are represented as a box-and-whisker plot, with bounds ranging from 25th to 75th percentile, the line representing the median, whiskers ranging from minimum to maximum values, and + indicating the mean. * P

Techniques Used: Mouse Assay, Isolation, Staining, Cell Culture, Enzyme-linked Immunosorbent Assay, Whisker Assay

Neutrophils and their associated inflammatory mediators are differentially regulated by IL-1R1 and IL-1RA during experimental fungal–associated allergic airway inflammation. C57BL/6 WT, IL-1 receptor–deficient ( Il1r1 –/– ), and IL-1 receptor antagonist–deficient ( Il1rn –/– ) mice were chronically exposed to A . fumigatus as described in the Methods. ( A and B ) Twenty-four hours after the last challenge, lung cells from ( A ) WT and Il1r1 –/– mice and from ( B ) WT and Il1rn –/– mice were isolated by bronchoalveolar lavage, enumerated, Fc-blocked, stained with a live/dead staining kit, and stained for neutrophils (CD45 + , CD11b + , Ly-6G + , Siglec F – ). The figures illustrate cumulative data from 2–3 independent studies ( n = 4–5 mice per group per study). ( C and D ) Twenty-four hours after the last challenge, right lungs were collected and enzymatically digested, and unfractionated lung cells were cocultured with A . fumigatus conidia for 24 hours at a cell/organism ratio of 1:1. IL-17A and IL-22 levels in lung digest cell culture supernatants from ( C ) WT and Il1r1 –/– mice and ( D ) WT and Il1rn –/– mice were quantified by MilliPlex or ELISA. The figures illustrate cumulative data from 2 independent studies ( n = 3 mice per group per study). ( E and F ) Samples obtained as described for C and D . G-CSF, CCL3, and CCL4 levels in lung digest cell culture supernatants from ( E ) WT and Il1r1 –/– mice and ( F ) WT and Il1rn –/– mice were quantified by MilliPlex. The figures illustrate cumulative data from 3 independent studies ( n = 3–5 mice per group per study). For all graphs, quantitative data are represented as a box-and-whisker plot, with bounds ranging from 25th to 75th percentile, the line representing the median, whiskers ranging from minimum to maximum values, and + indicating the mean. * P
Figure Legend Snippet: Neutrophils and their associated inflammatory mediators are differentially regulated by IL-1R1 and IL-1RA during experimental fungal–associated allergic airway inflammation. C57BL/6 WT, IL-1 receptor–deficient ( Il1r1 –/– ), and IL-1 receptor antagonist–deficient ( Il1rn –/– ) mice were chronically exposed to A . fumigatus as described in the Methods. ( A and B ) Twenty-four hours after the last challenge, lung cells from ( A ) WT and Il1r1 –/– mice and from ( B ) WT and Il1rn –/– mice were isolated by bronchoalveolar lavage, enumerated, Fc-blocked, stained with a live/dead staining kit, and stained for neutrophils (CD45 + , CD11b + , Ly-6G + , Siglec F – ). The figures illustrate cumulative data from 2–3 independent studies ( n = 4–5 mice per group per study). ( C and D ) Twenty-four hours after the last challenge, right lungs were collected and enzymatically digested, and unfractionated lung cells were cocultured with A . fumigatus conidia for 24 hours at a cell/organism ratio of 1:1. IL-17A and IL-22 levels in lung digest cell culture supernatants from ( C ) WT and Il1r1 –/– mice and ( D ) WT and Il1rn –/– mice were quantified by MilliPlex or ELISA. The figures illustrate cumulative data from 2 independent studies ( n = 3 mice per group per study). ( E and F ) Samples obtained as described for C and D . G-CSF, CCL3, and CCL4 levels in lung digest cell culture supernatants from ( E ) WT and Il1r1 –/– mice and ( F ) WT and Il1rn –/– mice were quantified by MilliPlex. The figures illustrate cumulative data from 3 independent studies ( n = 3–5 mice per group per study). For all graphs, quantitative data are represented as a box-and-whisker plot, with bounds ranging from 25th to 75th percentile, the line representing the median, whiskers ranging from minimum to maximum values, and + indicating the mean. * P

Techniques Used: Mouse Assay, Isolation, Staining, Cell Culture, Enzyme-linked Immunosorbent Assay, Whisker Assay

Cellular changes in whole lung during experimental fungal–associated allergic airway inflammation. C57BL/6 WT, IL-1 receptor–deficient ( Il1r1 –/– ), and IL-1 receptor antagonist–deficient ( Il1rn –/– ) mice were chronically exposed to A . fumigatus as in Methods. Twenty-four hours after the last challenge, right lungs were collected, enzymatically digested, Fc-blocked, stained with a live/dead staining kit, and stained for eosinophils (CD45 + , CD11b + , Siglec F + , Ly-6G – ), neutrophils (CD45 + , CD11b + , Ly-6G + ), CD4 + T cells (CD45 + , CD4 + , CD3 + ), γδ T cells (CD45 + , γδ TCR + , CD3 + ), and iNKT cells (TCRβ + , PBS-57 CD1d tetramer + ) in ( A ) WT and Il1r1 –/– mice and ( B ) WT and Il1rn –/– mice. The figures illustrate cumulative data from 2 independent studies ( n = 3–4 mice per group per study). For all graphs, quantitative data are represented as a box-and-whisker plot, with bounds ranging from 25th to 75th percentile, the line representing the median, whiskers ranging from minimum to maximum values, and + indicating the mean. * P
Figure Legend Snippet: Cellular changes in whole lung during experimental fungal–associated allergic airway inflammation. C57BL/6 WT, IL-1 receptor–deficient ( Il1r1 –/– ), and IL-1 receptor antagonist–deficient ( Il1rn –/– ) mice were chronically exposed to A . fumigatus as in Methods. Twenty-four hours after the last challenge, right lungs were collected, enzymatically digested, Fc-blocked, stained with a live/dead staining kit, and stained for eosinophils (CD45 + , CD11b + , Siglec F + , Ly-6G – ), neutrophils (CD45 + , CD11b + , Ly-6G + ), CD4 + T cells (CD45 + , CD4 + , CD3 + ), γδ T cells (CD45 + , γδ TCR + , CD3 + ), and iNKT cells (TCRβ + , PBS-57 CD1d tetramer + ) in ( A ) WT and Il1r1 –/– mice and ( B ) WT and Il1rn –/– mice. The figures illustrate cumulative data from 2 independent studies ( n = 3–4 mice per group per study). For all graphs, quantitative data are represented as a box-and-whisker plot, with bounds ranging from 25th to 75th percentile, the line representing the median, whiskers ranging from minimum to maximum values, and + indicating the mean. * P

Techniques Used: Mouse Assay, Staining, Whisker Assay

8) Product Images from "Respiratory Syncytial Virus (RSV) Infection in Elderly Mice Results in Altered Antiviral Gene Expression and Enhanced Pathology"

Article Title: Respiratory Syncytial Virus (RSV) Infection in Elderly Mice Results in Altered Antiviral Gene Expression and Enhanced Pathology

Journal: PLoS ONE

doi: 10.1371/journal.pone.0088764

Mucus secretion in response to rA2-L19F is altered in OPN-/- mice. WT C57BL/6 and OPN-/- mice were intranasally infection with a high dose of mucogenic rA2-L19F (1×10 6 pfu/mouse, n = 4) or a mock-inoculum, and at 5 and 8 dpi tissues were harvested. (A) Lung sections (5 µm) were stained with PAS per the manufacturer's instructions and counterstained with hematoxlin and eosin, then analyzed to calculate area of staining with ImageJ using Color Deconvolution plugin using single-stained customized vector settings. (B) The areas obtained through ImageJ analysis were related as a ratio of PAS-staining to hematoxylin staining, and displayed on a graph. A minimum of 10 fields of view were obtained at 200X per mouse and the images shown are a representation of the images collected. The experiment was performed in triplicate and data is displayed as means ±SEM.
Figure Legend Snippet: Mucus secretion in response to rA2-L19F is altered in OPN-/- mice. WT C57BL/6 and OPN-/- mice were intranasally infection with a high dose of mucogenic rA2-L19F (1×10 6 pfu/mouse, n = 4) or a mock-inoculum, and at 5 and 8 dpi tissues were harvested. (A) Lung sections (5 µm) were stained with PAS per the manufacturer's instructions and counterstained with hematoxlin and eosin, then analyzed to calculate area of staining with ImageJ using Color Deconvolution plugin using single-stained customized vector settings. (B) The areas obtained through ImageJ analysis were related as a ratio of PAS-staining to hematoxylin staining, and displayed on a graph. A minimum of 10 fields of view were obtained at 200X per mouse and the images shown are a representation of the images collected. The experiment was performed in triplicate and data is displayed as means ±SEM.

Techniques Used: Mouse Assay, Infection, Staining, Plasmid Preparation

OPN-/- mice are protected from RSV infections. WT C57BL/6 and OPN-/- mice were intranasally infection with a high dose of mucogenic rA2-L19F (1×10 6 pfu/mouse, n = 4) or a mock-inoculum, and at 5 and 8 dpi tissues were harvested. Total experiment was performed in triplicate, with data represented as means ±SEM. (A) Total lung RNA was analyzed with qRT-PCR for gene expression of RSV N and displayed as arbitrary relative units. (B) Plaques were obtained from the lung homogenates of infected mice at 5 and 8 dpi. (C) Lung sections (5 µm) were stained for the presence of RSV antigens using a polyclonal antibody and Alexa Fluor 555-secondary antibody and appears as red, while DAPI-stained nuclei appear blue. Images shown are representative of a minimum of 10 images collected at 200X magnification, all collected with identical exposure settings. (D) The number of RSV-positive cells was then calculated by ITCN ImageJ analysis, assuming differential diameters for quantifying the nuclei and individual cells. The percentage of RSV-positive cells is displayed as means ±SEM.
Figure Legend Snippet: OPN-/- mice are protected from RSV infections. WT C57BL/6 and OPN-/- mice were intranasally infection with a high dose of mucogenic rA2-L19F (1×10 6 pfu/mouse, n = 4) or a mock-inoculum, and at 5 and 8 dpi tissues were harvested. Total experiment was performed in triplicate, with data represented as means ±SEM. (A) Total lung RNA was analyzed with qRT-PCR for gene expression of RSV N and displayed as arbitrary relative units. (B) Plaques were obtained from the lung homogenates of infected mice at 5 and 8 dpi. (C) Lung sections (5 µm) were stained for the presence of RSV antigens using a polyclonal antibody and Alexa Fluor 555-secondary antibody and appears as red, while DAPI-stained nuclei appear blue. Images shown are representative of a minimum of 10 images collected at 200X magnification, all collected with identical exposure settings. (D) The number of RSV-positive cells was then calculated by ITCN ImageJ analysis, assuming differential diameters for quantifying the nuclei and individual cells. The percentage of RSV-positive cells is displayed as means ±SEM.

Techniques Used: Mouse Assay, Infection, Quantitative RT-PCR, Expressing, Staining

9) Product Images from "TNF receptors differentially signal and are differentially expressed and regulated in the human heart"

Article Title: TNF receptors differentially signal and are differentially expressed and regulated in the human heart

Journal: American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons

doi: 10.1111/j.1600-6143.2009.02831.x

(A). TNF-treated WT C57BL/6 cultures (a) but not TNF-treated TNFR1 KO cultures (b) show increased TUNEL-positive CM (open arrows) and VEC (arrows). In comparison, an increased TUNEL staining is seen in CM (arrowhead) and in VEC (arrow) in TNF-treated TNFR2 KO cultures (c). Apoptotic index (d) show statistically significant difference between cultures with the highest level of apoptotic cell death observed in TNF-treated TNFR2 KO cultures as compared to TNF-treated WT C57BL/6 (*p
Figure Legend Snippet: (A). TNF-treated WT C57BL/6 cultures (a) but not TNF-treated TNFR1 KO cultures (b) show increased TUNEL-positive CM (open arrows) and VEC (arrows). In comparison, an increased TUNEL staining is seen in CM (arrowhead) and in VEC (arrow) in TNF-treated TNFR2 KO cultures (c). Apoptotic index (d) show statistically significant difference between cultures with the highest level of apoptotic cell death observed in TNF-treated TNFR2 KO cultures as compared to TNF-treated WT C57BL/6 (*p

Techniques Used: TUNEL Assay, Staining

(A) . Untreated WT C57BL/6 cultures show positive staining for ASK1p-Ser967 and ASK1p-Thr845 in CM (open arrows) and in VEC (arrows) (a,b). Untreated TNFR1 KO cultures are positive for ASK1p-Ser967 (arrowhead) but not for ASK1p-Thr845 (c,d). In contrast, TNF-treated WT C57BL/6 cultures show a diminished ASK1p-Ser967 (g) but a strong signal for ASK1p-Thr845 in CM (open arrow) and in VEC (arrowhead) (h) while TNF-treated TNFR1 KO are negative (i,j). In contrast, CM (open arrow) and VEC (arrow) are strongly positive for ASK1p-Thr845 but not for ASK1p-Ser967 in TNF-treated TNFR2 KO cultures (k–l). (B). A strong signal for total Etk and phosphorylated Etk (Etkp) is seen in CM (open arrow) and in VEC (arrow) in untreated WT C57BL/6 (a,b) and in TNFR1 KO cultures (c,d), while untreated TNFR2 KO cultures are negative (e,f). TNF-treated WT C57BL/6 and TNFR1 KO cultures show Etk and Etkp in VEC (arrows) and in CM (open arrows) (g–j). In contrast, TNF-treated TNFR2 KO cultures are negative (k,l). Images are representative of 3 independent experiments with similar results (Original Mags, ×63).
Figure Legend Snippet: (A) . Untreated WT C57BL/6 cultures show positive staining for ASK1p-Ser967 and ASK1p-Thr845 in CM (open arrows) and in VEC (arrows) (a,b). Untreated TNFR1 KO cultures are positive for ASK1p-Ser967 (arrowhead) but not for ASK1p-Thr845 (c,d). In contrast, TNF-treated WT C57BL/6 cultures show a diminished ASK1p-Ser967 (g) but a strong signal for ASK1p-Thr845 in CM (open arrow) and in VEC (arrowhead) (h) while TNF-treated TNFR1 KO are negative (i,j). In contrast, CM (open arrow) and VEC (arrow) are strongly positive for ASK1p-Thr845 but not for ASK1p-Ser967 in TNF-treated TNFR2 KO cultures (k–l). (B). A strong signal for total Etk and phosphorylated Etk (Etkp) is seen in CM (open arrow) and in VEC (arrow) in untreated WT C57BL/6 (a,b) and in TNFR1 KO cultures (c,d), while untreated TNFR2 KO cultures are negative (e,f). TNF-treated WT C57BL/6 and TNFR1 KO cultures show Etk and Etkp in VEC (arrows) and in CM (open arrows) (g–j). In contrast, TNF-treated TNFR2 KO cultures are negative (k,l). Images are representative of 3 independent experiments with similar results (Original Mags, ×63).

Techniques Used: Staining

10) Product Images from "Resistance of Foxp3+ Regulatory T Cells to Nur77-Induced Apoptosis Promotes Allograft Survival"

Article Title: Resistance of Foxp3+ Regulatory T Cells to Nur77-Induced Apoptosis Promotes Allograft Survival

Journal: PLoS ONE

doi: 10.1371/journal.pone.0002321

Long-term acceptance of MHC mismatched cardiac allograft in Nur77Tg recipients. (a) BALB/c cardiac allografts were accepted long-term by Nur77Tg recipients, in contrast to acute rejection in WT C57BL/6 recipients (p
Figure Legend Snippet: Long-term acceptance of MHC mismatched cardiac allograft in Nur77Tg recipients. (a) BALB/c cardiac allografts were accepted long-term by Nur77Tg recipients, in contrast to acute rejection in WT C57BL/6 recipients (p

Techniques Used:

11) Product Images from "Complement activation on neutrophils initiates endothelial adhesion and extravasation"

Article Title: Complement activation on neutrophils initiates endothelial adhesion and extravasation

Journal: Molecular immunology

doi: 10.1016/j.molimm.2019.09.011

In vivo K/BxN serum administration triggers intravascular complement activation. A) Wild type (WT) C57BL/6 mice were injected i.v. with K/BxN serum. After 10 or 20 min, blood was collected and white blood cells stained for C3 fragment deposition and CD11b, CD11a, PSGL-1 expression on Gr-1 high cells. Quantitative assessment of C3, CD11b, CD11a, and PSGL-1 expression level on Gr-1 high /Ly-6G high . B) In other experiments, C4 −/− , FB −/− , FcγR −/− , and C5 null mice were injected with K/BxN serum i.v. for 10 min, blood was collected and stained for C3 fragment deposition on Gr-1 high cells. Histograms are representative of n = 4-9 animals per genotype.
Figure Legend Snippet: In vivo K/BxN serum administration triggers intravascular complement activation. A) Wild type (WT) C57BL/6 mice were injected i.v. with K/BxN serum. After 10 or 20 min, blood was collected and white blood cells stained for C3 fragment deposition and CD11b, CD11a, PSGL-1 expression on Gr-1 high cells. Quantitative assessment of C3, CD11b, CD11a, and PSGL-1 expression level on Gr-1 high /Ly-6G high . B) In other experiments, C4 −/− , FB −/− , FcγR −/− , and C5 null mice were injected with K/BxN serum i.v. for 10 min, blood was collected and stained for C3 fragment deposition on Gr-1 high cells. Histograms are representative of n = 4-9 animals per genotype.

Techniques Used: In Vivo, Activation Assay, Mouse Assay, Injection, Staining, Expressing

12) Product Images from "IL-33 promotes anemia during chronic inflammation by inhibiting differentiation of erythroid progenitors"

Article Title: IL-33 promotes anemia during chronic inflammation by inhibiting differentiation of erythroid progenitors

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20200164

The receptor for IL-33, ST2, is expressed preferentially on erythroid progenitors. (A) Representative flow cytometric expression of cytokine receptors on CFU-E and GMP in BM of healthy SKG mice. FMO, fluorescence-minus-one. Representative of two independent experiments. (B) Ratio of transcript counts for indicated cytokine receptor genes in CFU-E and GMP by RNA sequencing (mean and SEM of two biological replicates). Data derived from Haemosphere database. (C) Representative flow cytometric expression of ST2 receptor on indicated progenitors in BM of ST2 −/− mice and WT mice bred in same facility, both on C57BL/6 background. Representative of two independent experiments. (D) Representative image of flow cytometric expression of ST2 on cells of the erythroid lineage of healthy SKG mice from least mature (pre–CFU-E) to most mature. Ery A, erythroblast A (Ter119 + CD71 + FSC hi ); Ery B, erythroblast B (Ter119 + CD71 + FSC lo ); Ery C, erythroblast C (Ter119 + CD71 neg FSC lo ); ProE, proerythroblast (CD71 + Ter119 int ); RBC, Ter119 + CD71 neg FSC lo . Representative of two independent experiments.
Figure Legend Snippet: The receptor for IL-33, ST2, is expressed preferentially on erythroid progenitors. (A) Representative flow cytometric expression of cytokine receptors on CFU-E and GMP in BM of healthy SKG mice. FMO, fluorescence-minus-one. Representative of two independent experiments. (B) Ratio of transcript counts for indicated cytokine receptor genes in CFU-E and GMP by RNA sequencing (mean and SEM of two biological replicates). Data derived from Haemosphere database. (C) Representative flow cytometric expression of ST2 receptor on indicated progenitors in BM of ST2 −/− mice and WT mice bred in same facility, both on C57BL/6 background. Representative of two independent experiments. (D) Representative image of flow cytometric expression of ST2 on cells of the erythroid lineage of healthy SKG mice from least mature (pre–CFU-E) to most mature. Ery A, erythroblast A (Ter119 + CD71 + FSC hi ); Ery B, erythroblast B (Ter119 + CD71 + FSC lo ); Ery C, erythroblast C (Ter119 + CD71 neg FSC lo ); ProE, proerythroblast (CD71 + Ter119 int ); RBC, Ter119 + CD71 neg FSC lo . Representative of two independent experiments.

Techniques Used: Expressing, Mouse Assay, Fluorescence, RNA Sequencing Assay, Derivative Assay

Curdlan injection perturbs hematopoiesis in SKG and WT C57BL/6 mice. Data for A–H are from SKG mice injected with curdlan to induce SpA or littermate controls injected with PBS. (A) Scatterplots of clinical score (left), change in tarsal size (middle), and bodyweight (right) against neutrophil infiltration of the paws. Points represent individual mice, and solid lines represent linear regression model with 95% confidence intervals (dotted lines); values were pooled from three independent experiments. (B) Dot plot of blood Hgb concentration against change in weight. Red dots indicate mice with gross small intestinal inflammation (enteritis). Points represent individual mice, and solid line represents linear regression model with 95% confidence intervals (dotted lines); values were pooled from two independent experiments. (C) Representative flow cytometric images of lineage neg , Sca1 + , cKit + (LSK) stem and progenitor cells. HSCs: LSK, CD150 + , CD48 neg ; MPP2: LSK, CD150 + , CD48 + ; MPP3/4: LSK, CD150 neg , CD48 + . Figures show mean and SD of indicated populations among live BM cells, n = five to six per group. Graphs show absolute numbers of indicated cells derived from one femur and one tibia in SpA or healthy mice. Points represent individual mice with mean, Mann-Whitney U test, representative of four independent experiments. (D) Absolute numbers of BM progenitors derived from one femur and one tibia in SpA or healthy mice (points represent individual mice with mean, Mann-Whitney U test, representative of four independent experiments). (E) Absolute number of live BM cells derived from one femur and one tibia in indicated groups (points show individual mice with mean, Mann-Whitney U test, representative of four independent experiments). (F) Absolute number of mature (Ter119 + ) erythroid cells and neutrophils (NEUT) in indicated groups (points show individual mice with mean, Mann-Whitney U test, representative of four independent experiments). (G) Graph showing expression of surface markers in erythroid progenitor stages used to generate erythroid differentiation pathway graphs. Points represent mean and SD of scaled median fluorescence intensity values for n = five mice. Connecting line is locally weighted scatterplot smoothing (LOWESS) spline, representative of four independent experiments. (H) Absolute number of live cells from the spleen (left), frequency of erythroid progenitors (lineage neg , Sca1 neg , cKit + , CD41 neg , CD16/32 neg , CD105 + ) among live cells (middle) and absolute number of erythroid progenitors per spleen (right) in indicated mice (points show individual mice with mean, Mann-Whitney U test, representative of four independent experiments). (I) C57BL/6 WT littermate mice were injected with curdlan or PBS and culled after 1 wk to generate the data shown in I–L. (I) Frequency of indicated progenitors among live BM cells with curdlan or PBS (points show individual mice with mean, Mann-Whitney U test, representative of four independent experiments). (J) Absolute numbers of BM progenitors, mature erythroid cells (Ter119 + ), and neutrophils (NEUT) derived from one femur and one tibia in WT mice after injecting curdlan or PBS (points show individual mice with mean, Mann-Whitney U test, representative of four independent experiments). (K) Erythroid differentiation pathway for WT mice after injection of PBS or curdlan. Points represent mean with SD for n = five to six mice per group, and lines show LOWESS splines, Mann-Whitney U test, representative of four independent experiments. (L) Blood Hgb concentration in WT mice after injection of PBS or curdlan (points show individual mice with mean, Mann-Whitney U test, values pooled from two independent experiments). *, P
Figure Legend Snippet: Curdlan injection perturbs hematopoiesis in SKG and WT C57BL/6 mice. Data for A–H are from SKG mice injected with curdlan to induce SpA or littermate controls injected with PBS. (A) Scatterplots of clinical score (left), change in tarsal size (middle), and bodyweight (right) against neutrophil infiltration of the paws. Points represent individual mice, and solid lines represent linear regression model with 95% confidence intervals (dotted lines); values were pooled from three independent experiments. (B) Dot plot of blood Hgb concentration against change in weight. Red dots indicate mice with gross small intestinal inflammation (enteritis). Points represent individual mice, and solid line represents linear regression model with 95% confidence intervals (dotted lines); values were pooled from two independent experiments. (C) Representative flow cytometric images of lineage neg , Sca1 + , cKit + (LSK) stem and progenitor cells. HSCs: LSK, CD150 + , CD48 neg ; MPP2: LSK, CD150 + , CD48 + ; MPP3/4: LSK, CD150 neg , CD48 + . Figures show mean and SD of indicated populations among live BM cells, n = five to six per group. Graphs show absolute numbers of indicated cells derived from one femur and one tibia in SpA or healthy mice. Points represent individual mice with mean, Mann-Whitney U test, representative of four independent experiments. (D) Absolute numbers of BM progenitors derived from one femur and one tibia in SpA or healthy mice (points represent individual mice with mean, Mann-Whitney U test, representative of four independent experiments). (E) Absolute number of live BM cells derived from one femur and one tibia in indicated groups (points show individual mice with mean, Mann-Whitney U test, representative of four independent experiments). (F) Absolute number of mature (Ter119 + ) erythroid cells and neutrophils (NEUT) in indicated groups (points show individual mice with mean, Mann-Whitney U test, representative of four independent experiments). (G) Graph showing expression of surface markers in erythroid progenitor stages used to generate erythroid differentiation pathway graphs. Points represent mean and SD of scaled median fluorescence intensity values for n = five mice. Connecting line is locally weighted scatterplot smoothing (LOWESS) spline, representative of four independent experiments. (H) Absolute number of live cells from the spleen (left), frequency of erythroid progenitors (lineage neg , Sca1 neg , cKit + , CD41 neg , CD16/32 neg , CD105 + ) among live cells (middle) and absolute number of erythroid progenitors per spleen (right) in indicated mice (points show individual mice with mean, Mann-Whitney U test, representative of four independent experiments). (I) C57BL/6 WT littermate mice were injected with curdlan or PBS and culled after 1 wk to generate the data shown in I–L. (I) Frequency of indicated progenitors among live BM cells with curdlan or PBS (points show individual mice with mean, Mann-Whitney U test, representative of four independent experiments). (J) Absolute numbers of BM progenitors, mature erythroid cells (Ter119 + ), and neutrophils (NEUT) derived from one femur and one tibia in WT mice after injecting curdlan or PBS (points show individual mice with mean, Mann-Whitney U test, representative of four independent experiments). (K) Erythroid differentiation pathway for WT mice after injection of PBS or curdlan. Points represent mean with SD for n = five to six mice per group, and lines show LOWESS splines, Mann-Whitney U test, representative of four independent experiments. (L) Blood Hgb concentration in WT mice after injection of PBS or curdlan (points show individual mice with mean, Mann-Whitney U test, values pooled from two independent experiments). *, P

Techniques Used: Injection, Mouse Assay, Concentration Assay, Derivative Assay, MANN-WHITNEY, Expressing, Fluorescence

Association between IL-33 and erythroid progenitors. (A) Frequency of transcript counts for indicated cytokine receptor genes in indicated progenitor populations. Gene for the IL-33 receptor, ST2, is highlighted. Data derived from Haemosphere RNA-sequencing database of BM progenitors. Bars represent mean and SEM of two biological replicates. (B) Volcano plot comparing gene expression between GMP and CFU-E. The IL-33 receptor gene, Il1rl1 , is highlighted, along with critical erythroid ( Gata1 ) and myeloid ( Spi1 , PU.1) transcription factors. Data derived from comparison of two paired replicates from independent experiments in the Haemosphere database. (C) Frequency of transcript counts for Il1rl1 (encoding ST2) among indicated progenitor populations. RETIC, reticulocyte. Bars represent mean and SEM of two biological replicates from the Haemosphere database. (D) Representative images of flow cytometric expression of the ST2 receptor on indicated progenitor populations in BM of WT and ST2 −/− (ST2 KO) mice. ST2 −/− mice were on C57BL/6 background; WT mice were bred under the same conditions. HSCs: Lineage neg , Sca1 + , cKit + , CD150 + , CD48 − ; MPPs: LSK, CD150 − , CD48 + ; pre-MEG-E, premegakaryocyte-erythroid progenitor (Lineage neg , Sca1 − , cKit + , CD41 − , CD16/32 − , CD105 − , CD150 + ). Representative of two independent experiments.
Figure Legend Snippet: Association between IL-33 and erythroid progenitors. (A) Frequency of transcript counts for indicated cytokine receptor genes in indicated progenitor populations. Gene for the IL-33 receptor, ST2, is highlighted. Data derived from Haemosphere RNA-sequencing database of BM progenitors. Bars represent mean and SEM of two biological replicates. (B) Volcano plot comparing gene expression between GMP and CFU-E. The IL-33 receptor gene, Il1rl1 , is highlighted, along with critical erythroid ( Gata1 ) and myeloid ( Spi1 , PU.1) transcription factors. Data derived from comparison of two paired replicates from independent experiments in the Haemosphere database. (C) Frequency of transcript counts for Il1rl1 (encoding ST2) among indicated progenitor populations. RETIC, reticulocyte. Bars represent mean and SEM of two biological replicates from the Haemosphere database. (D) Representative images of flow cytometric expression of the ST2 receptor on indicated progenitor populations in BM of WT and ST2 −/− (ST2 KO) mice. ST2 −/− mice were on C57BL/6 background; WT mice were bred under the same conditions. HSCs: Lineage neg , Sca1 + , cKit + , CD150 + , CD48 − ; MPPs: LSK, CD150 − , CD48 + ; pre-MEG-E, premegakaryocyte-erythroid progenitor (Lineage neg , Sca1 − , cKit + , CD41 − , CD16/32 − , CD105 − , CD150 + ). Representative of two independent experiments.

Techniques Used: Derivative Assay, RNA Sequencing Assay, Expressing, Mouse Assay

IL-33 is produced in the BM niche and increased with inflammation. (A) Blood Hgb concentration measured in IL-33 −/− and WT mice (points show individual mice with mean, Mann-Whitney U test, representative of two independent experiments). IL-33 −/− mice backcrossed for eight generations onto same C57BL/6 colony from which WT mice derived. (B) Proportion of indicated BM progenitors among live BM cells (points show individual mice with mean, two-way ANOVA with Sidak’s multiple comparison test, representative of four independent experiments). (C) Expression of indicated genes by RT-qPCR in cells sorted by FACS from flushed (CD169 + macrophage [MP], neutrophil [NEUT], Ly6C + monocyte [MONO]) or crushed and digested (mesenchymal stromal/stem cells [MSC], endothelium [ENDO], and osteoblastic lineage cell [OBC]) bones of healthy SKG mice. Expression normalized to Hprt (mean and SEM of n = three independent experiments, n = two mice per group per experiment, unpaired t test). (D) Left: Representative flow cytometric images of citrine (GFP) expression in CD169 + F4/80 + BM macrophages in WT mice ±CD169 staining and in mice expressing citrine at the Il33 locus (IL-33 cit/cit ). IL-33 cit/cit mice backcrossed for eight generations to the same C57BL/6 colony from which WT mice derived (mean and SD, n = eight, representative of two independent experiments). Right: Absolute number of citrine + cells derived from one femur and one tibia of IL-33 cit/cit mice (points show individual mice with mean and SD, pooled from two independent experiments). (E) Concentration of IL-33 in BM plasma of littermate SKG mice before (day 0) and indicated time after curdlan injection (points show individual mice with mean and SD, one-way ANOVA with Tukey’s test, representative of two independent experiments). (F) Expression of indicated genes by RT-qPCR in indicated populations sorted by FACS as for C in healthy SKG mice and littermates with SpA. Expression normalized to Hprt (mean and SEM of n = three independent experiments, n = two mice per group per experiment, two-way ANOVA with Sidak’s multiple comparison test). *, P
Figure Legend Snippet: IL-33 is produced in the BM niche and increased with inflammation. (A) Blood Hgb concentration measured in IL-33 −/− and WT mice (points show individual mice with mean, Mann-Whitney U test, representative of two independent experiments). IL-33 −/− mice backcrossed for eight generations onto same C57BL/6 colony from which WT mice derived. (B) Proportion of indicated BM progenitors among live BM cells (points show individual mice with mean, two-way ANOVA with Sidak’s multiple comparison test, representative of four independent experiments). (C) Expression of indicated genes by RT-qPCR in cells sorted by FACS from flushed (CD169 + macrophage [MP], neutrophil [NEUT], Ly6C + monocyte [MONO]) or crushed and digested (mesenchymal stromal/stem cells [MSC], endothelium [ENDO], and osteoblastic lineage cell [OBC]) bones of healthy SKG mice. Expression normalized to Hprt (mean and SEM of n = three independent experiments, n = two mice per group per experiment, unpaired t test). (D) Left: Representative flow cytometric images of citrine (GFP) expression in CD169 + F4/80 + BM macrophages in WT mice ±CD169 staining and in mice expressing citrine at the Il33 locus (IL-33 cit/cit ). IL-33 cit/cit mice backcrossed for eight generations to the same C57BL/6 colony from which WT mice derived (mean and SD, n = eight, representative of two independent experiments). Right: Absolute number of citrine + cells derived from one femur and one tibia of IL-33 cit/cit mice (points show individual mice with mean and SD, pooled from two independent experiments). (E) Concentration of IL-33 in BM plasma of littermate SKG mice before (day 0) and indicated time after curdlan injection (points show individual mice with mean and SD, one-way ANOVA with Tukey’s test, representative of two independent experiments). (F) Expression of indicated genes by RT-qPCR in indicated populations sorted by FACS as for C in healthy SKG mice and littermates with SpA. Expression normalized to Hprt (mean and SEM of n = three independent experiments, n = two mice per group per experiment, two-way ANOVA with Sidak’s multiple comparison test). *, P

Techniques Used: Produced, Concentration Assay, Mouse Assay, MANN-WHITNEY, Derivative Assay, Expressing, Quantitative RT-PCR, FACS, Staining, Injection

IL-33 is implicated in suppression of erythropoiesis during chronic inflammation. (A) Schematic diagram showing injection of curdlan (CURD) or PBS in IL-33 −/− mice backcrossed for eight generations onto same C57BL/6 colony from which WT control mice derived, before culling after 1 wk to obtain data shown in B. (B) Frequency of CFU-E (left, middle) and GMP (right) among live BM cells. For CFU-Es, results are presented as pooled values from three independent experiments (left; points show individual mice with mean, one-way ANOVA with Tukey’s test) or as a summary of the same three independent experiments (middle) with mean and SEM ( n = four to six mice per group per experiment, unpaired t test). For GMP, points show individual mice with mean pooled from three independent experiments. Groups compared by one-way ANOVA with Tukey’s test. (C) Schematic diagram showing injection of SKG mice with recombinant murine IL-33, after injection of PBS or curdlan in littermate controls, before culling after 1 wk to obtain data shown in D, E, and G. (D) Frequency of indicated progenitors among live BM cells (points show individual mice with mean, one-way ANOVA with Tukey’s test, representative of three independent experiments). (E) Erythroid differentiation pathway (mean and SD for n = five to six mice per group with locally weighted scatterplot smoothing (LOWESS) spline, Mann-Whitney U test, representative of three independent experiments). (F) Frequency of indicated colonies from whole BM from healthy SKG mice in methylcellulose medium ±IL-33 after 10 d (mean and SD from n = three to four mice per group, two-way ANOVA with Sidak’s multiple comparison test, representative of two independent experiments). (G) Gene expression by RT-qPCR in FACS-sorted pre–CFU-Es from BM of healthy SKG mice and littermate controls injected with IL-33 (mean and SEM of n = three independent experiments, n = two mice per group per experiment, unpaired t test). (H) Schematic diagram showing injection of SKG mice with IL-33 or PBS in littermate controls over 4 wk to obtain data shown in I and J. (I) Frequency of erythroid (Ter119 + ) cells among live BM cells (points show individual mice with mean, Mann-Whitney U test, representative of two independent experiments). (J) Blood Hgb concentration (left) and RBC count (right). Points show individual mice with mean, Mann-Whitney U test, representative of two independent experiments. *, P
Figure Legend Snippet: IL-33 is implicated in suppression of erythropoiesis during chronic inflammation. (A) Schematic diagram showing injection of curdlan (CURD) or PBS in IL-33 −/− mice backcrossed for eight generations onto same C57BL/6 colony from which WT control mice derived, before culling after 1 wk to obtain data shown in B. (B) Frequency of CFU-E (left, middle) and GMP (right) among live BM cells. For CFU-Es, results are presented as pooled values from three independent experiments (left; points show individual mice with mean, one-way ANOVA with Tukey’s test) or as a summary of the same three independent experiments (middle) with mean and SEM ( n = four to six mice per group per experiment, unpaired t test). For GMP, points show individual mice with mean pooled from three independent experiments. Groups compared by one-way ANOVA with Tukey’s test. (C) Schematic diagram showing injection of SKG mice with recombinant murine IL-33, after injection of PBS or curdlan in littermate controls, before culling after 1 wk to obtain data shown in D, E, and G. (D) Frequency of indicated progenitors among live BM cells (points show individual mice with mean, one-way ANOVA with Tukey’s test, representative of three independent experiments). (E) Erythroid differentiation pathway (mean and SD for n = five to six mice per group with locally weighted scatterplot smoothing (LOWESS) spline, Mann-Whitney U test, representative of three independent experiments). (F) Frequency of indicated colonies from whole BM from healthy SKG mice in methylcellulose medium ±IL-33 after 10 d (mean and SD from n = three to four mice per group, two-way ANOVA with Sidak’s multiple comparison test, representative of two independent experiments). (G) Gene expression by RT-qPCR in FACS-sorted pre–CFU-Es from BM of healthy SKG mice and littermate controls injected with IL-33 (mean and SEM of n = three independent experiments, n = two mice per group per experiment, unpaired t test). (H) Schematic diagram showing injection of SKG mice with IL-33 or PBS in littermate controls over 4 wk to obtain data shown in I and J. (I) Frequency of erythroid (Ter119 + ) cells among live BM cells (points show individual mice with mean, Mann-Whitney U test, representative of two independent experiments). (J) Blood Hgb concentration (left) and RBC count (right). Points show individual mice with mean, Mann-Whitney U test, representative of two independent experiments. *, P

Techniques Used: Injection, Mouse Assay, Derivative Assay, Recombinant, MANN-WHITNEY, Expressing, Quantitative RT-PCR, FACS, Concentration Assay

13) Product Images from "Lipopolysaccharide suppresses IgE-mast cell mediated reactions"

Article Title: Lipopolysaccharide suppresses IgE-mast cell mediated reactions

Journal: Clinical and experimental allergy : journal of the British Society for Allergy and Clinical Immunology

doi: 10.1111/cea.13013

Temporal suppression of IgE-mediated reactions in C57BL/6 mice by LPS (A) experimental regime (B) Rectal temperature change from 0 to 60 min, (C) Hematocrit and (D) serum MCPT-1 levels at 60 min in LPS-treated WT C57BL6 mice following anti-IgE challenge. C57BL6 mice were i.p. injected with vehicle (+) or 1 mg/kg LPS and 24, 48 or 72 hours later received an i.v. injection of anti-IgE (20 μg) and evidence of shock response was assessed. Data shown for Vehicle (+) is of the 24-hour time point. Data represent mean ± SEM. N = 4–8 per group; Data were analyzed by one-way ANOVA and Tukey’s multiple comparison post-test *P
Figure Legend Snippet: Temporal suppression of IgE-mediated reactions in C57BL/6 mice by LPS (A) experimental regime (B) Rectal temperature change from 0 to 60 min, (C) Hematocrit and (D) serum MCPT-1 levels at 60 min in LPS-treated WT C57BL6 mice following anti-IgE challenge. C57BL6 mice were i.p. injected with vehicle (+) or 1 mg/kg LPS and 24, 48 or 72 hours later received an i.v. injection of anti-IgE (20 μg) and evidence of shock response was assessed. Data shown for Vehicle (+) is of the 24-hour time point. Data represent mean ± SEM. N = 4–8 per group; Data were analyzed by one-way ANOVA and Tukey’s multiple comparison post-test *P

Techniques Used: Mouse Assay, Injection

LPS-mediated suppression of IgE-mediated reactions in C57BL/6 mice is TLR4-dependent (A) Rectal temperature change from 0 to 60 min, (B) hemacrit and (C) serum MCPT-1 levels 1 h following anti-IgE stimulation in from Vehicle- and LPS-treated WT and Tlr4-deficient ( Tlr4 −/− ) C57BL6 mice. Data represent mean ± SEM. N = 5 and 7 per group; Data were analyzed by Student’s T-test.
Figure Legend Snippet: LPS-mediated suppression of IgE-mediated reactions in C57BL/6 mice is TLR4-dependent (A) Rectal temperature change from 0 to 60 min, (B) hemacrit and (C) serum MCPT-1 levels 1 h following anti-IgE stimulation in from Vehicle- and LPS-treated WT and Tlr4-deficient ( Tlr4 −/− ) C57BL6 mice. Data represent mean ± SEM. N = 5 and 7 per group; Data were analyzed by Student’s T-test.

Techniques Used: Mouse Assay

LPS suppresses IgE-mediated reactions in C57BL/6 mice (A) The experimental protocol is shown. Mice were i.p. injected with 0, 0.01, 0.1 or 1 mg/kg LPS, and 24 h later, were i.v. injected with 20 μg EM-95, and every mouse was subjected to peritoneal wash 1 h after EM-95 injection. The peritoneal cells were measured and analyzed by flow cytometry. MC were identified as c-Kit + , FcεRI + and ST2 + . (B) Rectal temperature change from 0 to 2 h after LPS i.p. injection. Serum TNF (C) and MCPT1 (D) concentration at 1 or 2 h after i.p. injection. Rectal temperature change from 0 to 60 min (F) and serum MCPT-1 levels in Vehicle- and LPS-treated WT C57BL6 mice following after EM-95 i.v. injection. (G). Representative dodt plot and histogram of FceRI expression on FSC low SSC high C-Kit + ST2+ peritoneal mast cells from Vehicle- (Upper panel) and LPS-treated (Lower panel) C57BL6 mice. Representative overlay histogram (H). and quantification of FceRI MFI on FSC low SSC high C-Kit + ST2 + peritoneal mast cells from Vehicle- and LPS-treated C57BL6 mice. Data represent mean ± SEM. n=4–10 per group; Data were analyzed by one-way or two-way ANOVA and Tukey’s multiple comparison post-test. *P
Figure Legend Snippet: LPS suppresses IgE-mediated reactions in C57BL/6 mice (A) The experimental protocol is shown. Mice were i.p. injected with 0, 0.01, 0.1 or 1 mg/kg LPS, and 24 h later, were i.v. injected with 20 μg EM-95, and every mouse was subjected to peritoneal wash 1 h after EM-95 injection. The peritoneal cells were measured and analyzed by flow cytometry. MC were identified as c-Kit + , FcεRI + and ST2 + . (B) Rectal temperature change from 0 to 2 h after LPS i.p. injection. Serum TNF (C) and MCPT1 (D) concentration at 1 or 2 h after i.p. injection. Rectal temperature change from 0 to 60 min (F) and serum MCPT-1 levels in Vehicle- and LPS-treated WT C57BL6 mice following after EM-95 i.v. injection. (G). Representative dodt plot and histogram of FceRI expression on FSC low SSC high C-Kit + ST2+ peritoneal mast cells from Vehicle- (Upper panel) and LPS-treated (Lower panel) C57BL6 mice. Representative overlay histogram (H). and quantification of FceRI MFI on FSC low SSC high C-Kit + ST2 + peritoneal mast cells from Vehicle- and LPS-treated C57BL6 mice. Data represent mean ± SEM. n=4–10 per group; Data were analyzed by one-way or two-way ANOVA and Tukey’s multiple comparison post-test. *P

Techniques Used: Mouse Assay, Injection, Flow Cytometry, Cytometry, Concentration Assay, Expressing

14) Product Images from "Toxoplasma gondii Ingests and Digests Host Cytosolic Proteins"

Article Title: Toxoplasma gondii Ingests and Digests Host Cytosolic Proteins

Journal: mBio

doi: 10.1128/mBio.01188-14

CPL contributes to the course of infection. (A) In vivo replication of PruΔ ku80LUC and PruΔ ku80LUC Δ cpl parasites assessed by bioluminescence imaging. Albino C57BL/6 mice (5 per group) were infected with the indicated doses of parasites by intraperitoneal injection. Mice were injected with d -luciferin, anesthetized, and imaged ventrally for 1 min. A cross indicates the point at which all mice became moribund and were humanely euthanized. (B) Pseudocolored images of bioluminescence from mice representative of PruΔ ku80LUC or PruΔ ku80LUC Δ cpl groups infected with 10 3 parasites. (C) Pseudocolored images of bioluminescence from mice representative of PruΔ ku80LUC or PruΔ ku80LUC Δ cpl groups infected with 10 6 parasites. (D) Mouse weight as an indicator of disease severity. Infected animals from the group inoculated with 10 3 or 10 6 parasites in panel A were weighed, and the values were plotted as the percentage of the initial weight over time. (E) Infection of IFN-γR −/− mice suggests a role for CPL in evasion of innate immunity. C57BL/6 WT or IFN-γR −/− mice (5 per group) were inoculated intraperitoneally with 10 4 PruΔ ku80LUC or PruΔ ku80LUC Δ cpl parasites and monitored for bioluminescence.
Figure Legend Snippet: CPL contributes to the course of infection. (A) In vivo replication of PruΔ ku80LUC and PruΔ ku80LUC Δ cpl parasites assessed by bioluminescence imaging. Albino C57BL/6 mice (5 per group) were infected with the indicated doses of parasites by intraperitoneal injection. Mice were injected with d -luciferin, anesthetized, and imaged ventrally for 1 min. A cross indicates the point at which all mice became moribund and were humanely euthanized. (B) Pseudocolored images of bioluminescence from mice representative of PruΔ ku80LUC or PruΔ ku80LUC Δ cpl groups infected with 10 3 parasites. (C) Pseudocolored images of bioluminescence from mice representative of PruΔ ku80LUC or PruΔ ku80LUC Δ cpl groups infected with 10 6 parasites. (D) Mouse weight as an indicator of disease severity. Infected animals from the group inoculated with 10 3 or 10 6 parasites in panel A were weighed, and the values were plotted as the percentage of the initial weight over time. (E) Infection of IFN-γR −/− mice suggests a role for CPL in evasion of innate immunity. C57BL/6 WT or IFN-γR −/− mice (5 per group) were inoculated intraperitoneally with 10 4 PruΔ ku80LUC or PruΔ ku80LUC Δ cpl parasites and monitored for bioluminescence.

Techniques Used: Infection, In Vivo, Imaging, Mouse Assay, Injection

Parasites deficient in CPL and GRA2 are virulence attenuated. (A) CPL and GRA2 contribute synergistically to virulence. One thousand parasites of each strain were used to infect outbred CD-1 mice by subcutaneous injection. Data are combined from two independent experiments, each with 5 mice per group. (B) Pru strain parasites deficient in CPL are virulence attenuated. The indicated doses of PruΔ ku80LUC and PruΔ ku80LUC Δ cpl parasites were injected into C57BL/6 mice intraperitoneally. Data are combined from two independent experiments, each with 5 mice per group.
Figure Legend Snippet: Parasites deficient in CPL and GRA2 are virulence attenuated. (A) CPL and GRA2 contribute synergistically to virulence. One thousand parasites of each strain were used to infect outbred CD-1 mice by subcutaneous injection. Data are combined from two independent experiments, each with 5 mice per group. (B) Pru strain parasites deficient in CPL are virulence attenuated. The indicated doses of PruΔ ku80LUC and PruΔ ku80LUC Δ cpl parasites were injected into C57BL/6 mice intraperitoneally. Data are combined from two independent experiments, each with 5 mice per group.

Techniques Used: Mouse Assay, Injection

15) Product Images from "IL-23-Independent IL-17 Production Regulates Intestinal Epithelial Permeability"

Article Title: IL-23-Independent IL-17 Production Regulates Intestinal Epithelial Permeability

Journal: Immunity

doi: 10.1016/j.immuni.2015.09.003

Dysregulation of occludin cellular localization in the absence of IL-17A following DSS induced injury (A) Colonic tissues were analyzed by RT PCR for the tight junction proteins ZO-1 and occludin mRNA at day 3 and day 7 after DSS treatment. No difference in ZO-1 or occludin message between WT or Il-17 −/− mice. (B) C57BL/6 mice were subcutaneously administered with either control IgG or anti-IL-17A antibody at 20mg/kg 2 days before DSS treatment. Immunofluorescence images of occludin (green), f-actin (red), DNA (blue) of distal colon segments 3 days after DSS. The fluorescent images depict cross sections of the intestinal crypts in the distal colon with the apical surface of the cell oriented toward the lumen (L). The third column represents a magnified image from the white box in the second column. ( C ) Caco-2 cells were plated on Poly-L-lysine coated coverslips and treated with recombinant human TNFα (10ng/mL) or TNFα (10ng/mL) + recombinant human IL-17A (10ng/mL) and cultured for 24 hrs. Immunofluorescence images of occludin (green) and DNA (blue). The bottom row represents a magnified image from the corresponding white box in the first row.
Figure Legend Snippet: Dysregulation of occludin cellular localization in the absence of IL-17A following DSS induced injury (A) Colonic tissues were analyzed by RT PCR for the tight junction proteins ZO-1 and occludin mRNA at day 3 and day 7 after DSS treatment. No difference in ZO-1 or occludin message between WT or Il-17 −/− mice. (B) C57BL/6 mice were subcutaneously administered with either control IgG or anti-IL-17A antibody at 20mg/kg 2 days before DSS treatment. Immunofluorescence images of occludin (green), f-actin (red), DNA (blue) of distal colon segments 3 days after DSS. The fluorescent images depict cross sections of the intestinal crypts in the distal colon with the apical surface of the cell oriented toward the lumen (L). The third column represents a magnified image from the white box in the second column. ( C ) Caco-2 cells were plated on Poly-L-lysine coated coverslips and treated with recombinant human TNFα (10ng/mL) or TNFα (10ng/mL) + recombinant human IL-17A (10ng/mL) and cultured for 24 hrs. Immunofluorescence images of occludin (green) and DNA (blue). The bottom row represents a magnified image from the corresponding white box in the first row.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Mouse Assay, Immunofluorescence, Recombinant, Cell Culture

16) Product Images from "Platelet CD36 promotes thrombosis by activating redox sensor ERK5 in hyperlipidemic conditions"

Article Title: Platelet CD36 promotes thrombosis by activating redox sensor ERK5 in hyperlipidemic conditions

Journal: Blood

doi: 10.1182/blood-2016-11-750133

Platelet ERK5 promotes integrin activation, aggregation, and accumulation under shear by oxLDL-CD36 signaling. Washed human platelets (3 × 10 8 /mL) were pretreated with DMSO or BIX 02188 (10 µM) before stimulation with oxLDL (50 µg/mL). Integrin activation was detected with Alexa Fluor 647-labeled fibrinogen (15 µg/mL) (n = 7) (A), and α granule secretion was detected by PE-labeled P-selectin antibody (n = 7) (B) using flow cytometry. (C) Platelet aggregation over time was measured after washed human platelets (3 × 10 8 /mL) were preincubated with DMSO or BIX 02188 (10 µM) before stimulating with oxLDL (50 µg/mL) (n = 3). (D) Platelet aggregation was measured after gel-filtered mouse platelets (3 × 10 8 /mL) were pretreated with oxLDL (50 µg/mL) at room temperature (RT), before stimulating with 0.2 µM ADP (n = 3). (E) Platelet adhesion over time on immobilized collagen was measured after whole blood from WT C57Bl/6 (n = 5), CD36 null (n = 5), ERK5 flox/flox (n = 6), and ERK5 flox /PF4-cre + (n = 6) mice were stimulated with PBS or oxLDL (50 µg/mL). Data represented as mean ± SEM. P value determined by 2-tailed Student t test (C-D) and 1-way ANOVA with Tukey’s post hoc analysis (A-B), between WT and CD36-null (E), and between ERK5 flox/flox and ERK5 flox /PF4-cre + (E).
Figure Legend Snippet: Platelet ERK5 promotes integrin activation, aggregation, and accumulation under shear by oxLDL-CD36 signaling. Washed human platelets (3 × 10 8 /mL) were pretreated with DMSO or BIX 02188 (10 µM) before stimulation with oxLDL (50 µg/mL). Integrin activation was detected with Alexa Fluor 647-labeled fibrinogen (15 µg/mL) (n = 7) (A), and α granule secretion was detected by PE-labeled P-selectin antibody (n = 7) (B) using flow cytometry. (C) Platelet aggregation over time was measured after washed human platelets (3 × 10 8 /mL) were preincubated with DMSO or BIX 02188 (10 µM) before stimulating with oxLDL (50 µg/mL) (n = 3). (D) Platelet aggregation was measured after gel-filtered mouse platelets (3 × 10 8 /mL) were pretreated with oxLDL (50 µg/mL) at room temperature (RT), before stimulating with 0.2 µM ADP (n = 3). (E) Platelet adhesion over time on immobilized collagen was measured after whole blood from WT C57Bl/6 (n = 5), CD36 null (n = 5), ERK5 flox/flox (n = 6), and ERK5 flox /PF4-cre + (n = 6) mice were stimulated with PBS or oxLDL (50 µg/mL). Data represented as mean ± SEM. P value determined by 2-tailed Student t test (C-D) and 1-way ANOVA with Tukey’s post hoc analysis (A-B), between WT and CD36-null (E), and between ERK5 flox/flox and ERK5 flox /PF4-cre + (E).

Techniques Used: Activation Assay, Labeling, Flow Cytometry, Cytometry, Mouse Assay

17) Product Images from "Histone/protein deacetylase 11 targeting promotes Foxp3+ Treg function"

Article Title: Histone/protein deacetylase 11 targeting promotes Foxp3+ Treg function

Journal: Scientific Reports

doi: 10.1038/s41598-017-09211-3

Effects of pharmacologic inhibition of HDAC11 on Treg function and allograft survival. ( A ) Testing of the in vitro effects of the HDAC11i, JB3-22, on WT, Hdac6−/− and HDAC11−/− Treg suppressive function. Upper 3 rows show baseline function of WT vs. HDAC6−/− and HDAC11−/− Tregs in the presence of DMSO carrier alone; proliferation of CFSE-labeled conventional T cells is shown in each panel. The lower 3 rows show the effects of HDAC11i (0.1 µM) on Treg function using the same cell types as tested above. The point is that HDAC11i further increases the suppressive function of WT and HDAC6−/− Tregs, but does not have any additional effects on the suppressive function of HDAC11−/− Treg cells. Data are representative of 3 separate assays. ( B ) Effects of HDAC11i (JB3-22, (10 mg/kg/d, 14 d, i.p.) plus low-dose RPM (0.2 mg/kg/d, 14 d) in the BALB/c- > C57BL/6 cardiac allograft model. Control mice receiving low dose RPM rejected their allografts within 2–3 weeks, but cardiac allografts were maintained long-term ( > 100 days, p
Figure Legend Snippet: Effects of pharmacologic inhibition of HDAC11 on Treg function and allograft survival. ( A ) Testing of the in vitro effects of the HDAC11i, JB3-22, on WT, Hdac6−/− and HDAC11−/− Treg suppressive function. Upper 3 rows show baseline function of WT vs. HDAC6−/− and HDAC11−/− Tregs in the presence of DMSO carrier alone; proliferation of CFSE-labeled conventional T cells is shown in each panel. The lower 3 rows show the effects of HDAC11i (0.1 µM) on Treg function using the same cell types as tested above. The point is that HDAC11i further increases the suppressive function of WT and HDAC6−/− Tregs, but does not have any additional effects on the suppressive function of HDAC11−/− Treg cells. Data are representative of 3 separate assays. ( B ) Effects of HDAC11i (JB3-22, (10 mg/kg/d, 14 d, i.p.) plus low-dose RPM (0.2 mg/kg/d, 14 d) in the BALB/c- > C57BL/6 cardiac allograft model. Control mice receiving low dose RPM rejected their allografts within 2–3 weeks, but cardiac allografts were maintained long-term ( > 100 days, p

Techniques Used: Inhibition, In Vitro, Labeling, Mouse Assay

18) Product Images from "Batf3 deficiency is not critical for the generation of CD8α+ dendritic cells"

Article Title: Batf3 deficiency is not critical for the generation of CD8α+ dendritic cells

Journal: Immunobiology

doi: 10.1016/j.imbio.2014.10.019

Quantitation of gB DNA and LAT RNA in trigeminal ganglia of HSV-1 latently-infected mice Wild-type (WT) C57BL/6 or Batf3 −/− mice were ocularly-infected with HSV-1 strain McKrae. On day 28 PI, TG were harvested from latently infected mice. Quantitative PCR and RT-PCR was performed on each individual mouse TG. In each experiment, an estimated relative copy number of the HSV-1 gB (for viral DNA) and LAT (for viral RNA) were calculated using standard curves generated from pGem-gB1 and pGem5317, respectively. Briefly, DNA template was serially diluted 10-fold such that 5 μl contained from 10 3 to 10 11 copies of gB, then subjected to TaqMan PCR with the same set of primers. By comparing the normalized threshold cycle of each sample to the threshold cycle of the standard, the copy number for each reaction was determined. GAPDH expression was used to normalize the relative expression of viral (gB) DNA and LAT RNA in the TG. Each data point represents the mean ± SEM from 10 TG. Panels: A) gB DNA; and B) LAT RNA.
Figure Legend Snippet: Quantitation of gB DNA and LAT RNA in trigeminal ganglia of HSV-1 latently-infected mice Wild-type (WT) C57BL/6 or Batf3 −/− mice were ocularly-infected with HSV-1 strain McKrae. On day 28 PI, TG were harvested from latently infected mice. Quantitative PCR and RT-PCR was performed on each individual mouse TG. In each experiment, an estimated relative copy number of the HSV-1 gB (for viral DNA) and LAT (for viral RNA) were calculated using standard curves generated from pGem-gB1 and pGem5317, respectively. Briefly, DNA template was serially diluted 10-fold such that 5 μl contained from 10 3 to 10 11 copies of gB, then subjected to TaqMan PCR with the same set of primers. By comparing the normalized threshold cycle of each sample to the threshold cycle of the standard, the copy number for each reaction was determined. GAPDH expression was used to normalize the relative expression of viral (gB) DNA and LAT RNA in the TG. Each data point represents the mean ± SEM from 10 TG. Panels: A) gB DNA; and B) LAT RNA.

Techniques Used: Quantitation Assay, Infection, Mouse Assay, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Generated, Polymerase Chain Reaction, Expressing

19) Product Images from "MHCII glycosylation modulates Bacteroides fragilis carbohydrate antigen presentation"

Article Title: MHCII glycosylation modulates Bacteroides fragilis carbohydrate antigen presentation

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20100508

Native complex N-glycans are required for PSA-driven T cell activation in vitro. (A) BMDCs were cultured in the presence or absence of CS (red) and KF (blue) for 3 d, and then analyzed by PHA-L, PHA-E, WGA, and Con A lectin flow cytometry to detect N-glycans. Representative histograms and n = 4 MFI analyses are shown. (B) BMDCs were cultured for 8 d, probed with CD11c mAb, and analyzed by flow cytometry to verify proper DC cell differentiation. Representative histogram is shown. (C) BMDCs were incubated with nonfluorescent intact DQ-OVA protein for 24 h to allow uptake and processing. Cells were then analyzed for fluorescence, which is indicative of endocytosis and cleavage to peptides. Representative histograms are shown. n = 4 for MFI analyses. (D) BMDC maturation upon exposure to antigen was measured by surface staining of MHCII 24 h after stimulation with OVA peptide and LPS (O+L), LPS alone, or PSA. Representative histograms shown. (E) BMDCs grown with and without CS and KF were pulsed with either DQ-OVA (OVA), OVAp+LPS (O+L), or LPS alone for 24 h, after which untreated fresh naive CD4 + T cells isolated from OT-II animals were added. n = 3 per condition, per antigen. (F) As with OVAp, BMDCs were grown with CS and KF and pulsed with PSA, Sp1, or B. fragilis capsule (Caps) for 24 h. Next, naive WT C57BL/6 CD4 + T cells were added to allow activation to occur. n = 3 per condition. (G) TLR2 activation by PSA, a necessary step in GlyAg-mediated T cell activation ( Wang et al., 2006 ), and a control agonist Pam3Cysk4 (P3C) was measured in untreated (UNT), CS-treated, or KF-treated TLR2 + HEK293 cells. Bars represent background-subtracted values. n = 3 per condition. All bar graphs show the mean ± SEM.
Figure Legend Snippet: Native complex N-glycans are required for PSA-driven T cell activation in vitro. (A) BMDCs were cultured in the presence or absence of CS (red) and KF (blue) for 3 d, and then analyzed by PHA-L, PHA-E, WGA, and Con A lectin flow cytometry to detect N-glycans. Representative histograms and n = 4 MFI analyses are shown. (B) BMDCs were cultured for 8 d, probed with CD11c mAb, and analyzed by flow cytometry to verify proper DC cell differentiation. Representative histogram is shown. (C) BMDCs were incubated with nonfluorescent intact DQ-OVA protein for 24 h to allow uptake and processing. Cells were then analyzed for fluorescence, which is indicative of endocytosis and cleavage to peptides. Representative histograms are shown. n = 4 for MFI analyses. (D) BMDC maturation upon exposure to antigen was measured by surface staining of MHCII 24 h after stimulation with OVA peptide and LPS (O+L), LPS alone, or PSA. Representative histograms shown. (E) BMDCs grown with and without CS and KF were pulsed with either DQ-OVA (OVA), OVAp+LPS (O+L), or LPS alone for 24 h, after which untreated fresh naive CD4 + T cells isolated from OT-II animals were added. n = 3 per condition, per antigen. (F) As with OVAp, BMDCs were grown with CS and KF and pulsed with PSA, Sp1, or B. fragilis capsule (Caps) for 24 h. Next, naive WT C57BL/6 CD4 + T cells were added to allow activation to occur. n = 3 per condition. (G) TLR2 activation by PSA, a necessary step in GlyAg-mediated T cell activation ( Wang et al., 2006 ), and a control agonist Pam3Cysk4 (P3C) was measured in untreated (UNT), CS-treated, or KF-treated TLR2 + HEK293 cells. Bars represent background-subtracted values. n = 3 per condition. All bar graphs show the mean ± SEM.

Techniques Used: Activation Assay, In Vitro, Cell Culture, Whole Genome Amplification, Flow Cytometry, Cytometry, Cell Differentiation, Incubation, Fluorescence, Staining, Isolation

Native complex N-glycans are required for PSA-driven immune responses in vivo. (A) WT C57BL/6 animals ( n = 8) were treated daily for 1, 2, or 3 d with 250 µg of KF, and then sacrificed to harvest splenocytes. Cells were stained for native complex N-glycans using PHA-L. Representative histogram shown. (B) WT mice ( n = 3 per group) were treated with KF or PBS for 3 d, as in A. Animals were then sensitized with 100 µg PSA. Splenocytes were harvested 6 d later, stained for IFN-γ and CD4, and analyzed by flow cytometry. (C) KF- or PBS-pretreated mice ( n = 6) were sensitized with 100 µg OVA and sterile cecal content adjuvant. 4 d later, CD4 + T cells were isolated and restimulated ex vivo with normally glycosylated APCs and OVA ( n = 6). (D) Animals ( n = 6) were treated as in C, only with 100 µg of PSA rather than OVA. The PSA-specific recall ( n = 18) was measured after ex vivo restimulation with normally glycosylated APCs and PSA. (E) Animals ( n = 6) were treated as in D, only with the additional adoptive transfer of 5 × 10 6 fresh normally glycosylated APCs at the time of PSA injection ( n = 12). For panels C–E, data were normalized to the antigen-sensitized animals without KF. All bar graphs show the mean ± SEM.
Figure Legend Snippet: Native complex N-glycans are required for PSA-driven immune responses in vivo. (A) WT C57BL/6 animals ( n = 8) were treated daily for 1, 2, or 3 d with 250 µg of KF, and then sacrificed to harvest splenocytes. Cells were stained for native complex N-glycans using PHA-L. Representative histogram shown. (B) WT mice ( n = 3 per group) were treated with KF or PBS for 3 d, as in A. Animals were then sensitized with 100 µg PSA. Splenocytes were harvested 6 d later, stained for IFN-γ and CD4, and analyzed by flow cytometry. (C) KF- or PBS-pretreated mice ( n = 6) were sensitized with 100 µg OVA and sterile cecal content adjuvant. 4 d later, CD4 + T cells were isolated and restimulated ex vivo with normally glycosylated APCs and OVA ( n = 6). (D) Animals ( n = 6) were treated as in C, only with 100 µg of PSA rather than OVA. The PSA-specific recall ( n = 18) was measured after ex vivo restimulation with normally glycosylated APCs and PSA. (E) Animals ( n = 6) were treated as in D, only with the additional adoptive transfer of 5 × 10 6 fresh normally glycosylated APCs at the time of PSA injection ( n = 12). For panels C–E, data were normalized to the antigen-sensitized animals without KF. All bar graphs show the mean ± SEM.

Techniques Used: In Vivo, Staining, Mouse Assay, Flow Cytometry, Cytometry, Isolation, Ex Vivo, Adoptive Transfer Assay, Injection

20) Product Images from "Differential Functions of Tumor Necrosis Factor Receptor 1 and 2 Signaling in Ischemia-Mediated Arteriogenesis and Angiogenesis"

Article Title: Differential Functions of Tumor Necrosis Factor Receptor 1 and 2 Signaling in Ischemia-Mediated Arteriogenesis and Angiogenesis

Journal: The American Journal of Pathology

doi: 10.2353/ajpath.2006.060603

Critical roles of TNFR2 in the recovery of hindlimb perfusion after injury. a: A schematic diagram for the distinct signaling pathways mediated by TNFR1 and TNFR2. TNF through TNFR1 activates FADD-caspase-8-dependent (not shown in the figure) and ASK1/JNK-dependent apoptotic pathways. TNF activates Bmx/Etk survival and angiogenic signaling through TNFR2. b: Ischemic hindlimb model was performed. Blood flow of ischemic and nonischemic limb were measured on gastrocnemius muscle at 30 minutes, 3 days, 2 weeks, and 4 weeks after surgery by using the PeriFlux system with the laser Doppler perfusion measurement (LDPM) unit. Tissues were harvested on day 28 for immunohistochemistry. c and d: TNFR2-KO mice showed severe phenotype in clinical score ( c ) leading to necrosis of limb ( d ). e: TNFR2-KO mice show reduced whereas TNFR1-KO mice show augmented recovery of limb perfusion compared with normal C57BL/6 mice [ratio of perfusion unit from nonischemia (left) to ischemia (right) are shown]. Number for each strain is shown in parentheses. Data are mean ± SEM, * P
Figure Legend Snippet: Critical roles of TNFR2 in the recovery of hindlimb perfusion after injury. a: A schematic diagram for the distinct signaling pathways mediated by TNFR1 and TNFR2. TNF through TNFR1 activates FADD-caspase-8-dependent (not shown in the figure) and ASK1/JNK-dependent apoptotic pathways. TNF activates Bmx/Etk survival and angiogenic signaling through TNFR2. b: Ischemic hindlimb model was performed. Blood flow of ischemic and nonischemic limb were measured on gastrocnemius muscle at 30 minutes, 3 days, 2 weeks, and 4 weeks after surgery by using the PeriFlux system with the laser Doppler perfusion measurement (LDPM) unit. Tissues were harvested on day 28 for immunohistochemistry. c and d: TNFR2-KO mice showed severe phenotype in clinical score ( c ) leading to necrosis of limb ( d ). e: TNFR2-KO mice show reduced whereas TNFR1-KO mice show augmented recovery of limb perfusion compared with normal C57BL/6 mice [ratio of perfusion unit from nonischemia (left) to ischemia (right) are shown]. Number for each strain is shown in parentheses. Data are mean ± SEM, * P

Techniques Used: Flow Cytometry, Immunohistochemistry, Mouse Assay

Characterization of infiltrated immune cells, tissue necrosis/apoptosis, and cellular proliferation in TNFR1-KO and TNFR2-KO mice in response to ischemia. C57BL/6, TNFR1-KO, and TNFR2-KO mice were subjected to ischemia ligation, and tissues were harvested at indicated times. Tissue infiltrates, apoptosis, and cellular proliferation in gastrocnemius were determined by immunohistochemistry with respective markers. a – c: Recruitment of macrophages/lymphocytes in response to ischemia as determined by anti-F4/80 and anti-CD3 antibody, respectively. Representative images of nonischemic and ischemic hindlimbs in C57BL/6 on day 3 are shown in a in which macrophages and lymphocytes are indicated by arrows . F4/80- and CD3-positive cells were counted as number of infiltration/mm 2 muscle area in C57BL/6, TNFR1-KO, and TNFR2-KO mice on day 3 after ischemia ( b , c ). d- - f: Cellular proliferation in nonischemic and ischemic tissues (day 28) was determined by PCNA staining. d: PCNA-positive capillaries (CD31-positive) and muscle fibers are indicated by arrows and arrowhead , respectively. e: Representative images of nonischemic and ischemic hindlimbs in C57BL/6, TNFR1-KO, and TNFR2-KO mice are shown. f: PCNA-positive cells were counted as number/mm 2 muscle area. f: PCNA-positive cells were counted as number/mm 2 muscle area. Data from different mice groups are shown in graphics and n = 4 for each strain. * P
Figure Legend Snippet: Characterization of infiltrated immune cells, tissue necrosis/apoptosis, and cellular proliferation in TNFR1-KO and TNFR2-KO mice in response to ischemia. C57BL/6, TNFR1-KO, and TNFR2-KO mice were subjected to ischemia ligation, and tissues were harvested at indicated times. Tissue infiltrates, apoptosis, and cellular proliferation in gastrocnemius were determined by immunohistochemistry with respective markers. a – c: Recruitment of macrophages/lymphocytes in response to ischemia as determined by anti-F4/80 and anti-CD3 antibody, respectively. Representative images of nonischemic and ischemic hindlimbs in C57BL/6 on day 3 are shown in a in which macrophages and lymphocytes are indicated by arrows . F4/80- and CD3-positive cells were counted as number of infiltration/mm 2 muscle area in C57BL/6, TNFR1-KO, and TNFR2-KO mice on day 3 after ischemia ( b , c ). d- - f: Cellular proliferation in nonischemic and ischemic tissues (day 28) was determined by PCNA staining. d: PCNA-positive capillaries (CD31-positive) and muscle fibers are indicated by arrows and arrowhead , respectively. e: Representative images of nonischemic and ischemic hindlimbs in C57BL/6, TNFR1-KO, and TNFR2-KO mice are shown. f: PCNA-positive cells were counted as number/mm 2 muscle area. f: PCNA-positive cells were counted as number/mm 2 muscle area. Data from different mice groups are shown in graphics and n = 4 for each strain. * P

Techniques Used: Mouse Assay, Ligation, Immunohistochemistry, Staining

Ischemia induces TNFR2-specific signaling. a: C57BL/6 ( n = 3) mice were subjected to hindlimb ischemia, and hindlimbs were harvested on days 0, 3, 14, and 28 after surgery as indicated. Gene expression of TNFR1, TNFR2, TRAF2, and Bmx/Etk were determined by qRT-PCR. 18S rRNA was used for normalization. Fold induction (left/right) is shown. b and c: Day 3 samples were used for protein analyses. TNFR1, TNFR2, TRAF2, and Bmx/Etk, as well as Bmx/Etk phosphorylation (pY40), were determined by Western blot with respective antibodies. β-Tubulin was used as a control. Relative levels of pY40 and Bmx expression are shown, with ischemic WT as 1.0. Similar results were obtained in an additional five mice from two independent experiments (total n = 6). d: TNFR2-TRAF2 complexes in the lower limb tissues were determined by immunoprecipitation with anti-TRAF2 or normal rabbit serum (NRS) followed by Western blot with anti-TNFR2 antibody. TRAF2 in the immunoprecipitates was determined by Western blot with anti-TRAF2 antibody. Similar results were obtained from two additional experiments. e: TNFR2 was induced in vascular endothelium. TNFR2 protein in the frozen sections of nonischemic and ischemic (day 3) lower limb were detected by immunohistochemistry with anti-TNFR2 antibody. Capillaries (CD31-positive) found to be TNFR2-positive or -negative are indicated by arrows and arrowhead , respectively.
Figure Legend Snippet: Ischemia induces TNFR2-specific signaling. a: C57BL/6 ( n = 3) mice were subjected to hindlimb ischemia, and hindlimbs were harvested on days 0, 3, 14, and 28 after surgery as indicated. Gene expression of TNFR1, TNFR2, TRAF2, and Bmx/Etk were determined by qRT-PCR. 18S rRNA was used for normalization. Fold induction (left/right) is shown. b and c: Day 3 samples were used for protein analyses. TNFR1, TNFR2, TRAF2, and Bmx/Etk, as well as Bmx/Etk phosphorylation (pY40), were determined by Western blot with respective antibodies. β-Tubulin was used as a control. Relative levels of pY40 and Bmx expression are shown, with ischemic WT as 1.0. Similar results were obtained in an additional five mice from two independent experiments (total n = 6). d: TNFR2-TRAF2 complexes in the lower limb tissues were determined by immunoprecipitation with anti-TRAF2 or normal rabbit serum (NRS) followed by Western blot with anti-TNFR2 antibody. TRAF2 in the immunoprecipitates was determined by Western blot with anti-TRAF2 antibody. Similar results were obtained from two additional experiments. e: TNFR2 was induced in vascular endothelium. TNFR2 protein in the frozen sections of nonischemic and ischemic (day 3) lower limb were detected by immunohistochemistry with anti-TNFR2 antibody. Capillaries (CD31-positive) found to be TNFR2-positive or -negative are indicated by arrows and arrowhead , respectively.

Techniques Used: Mouse Assay, Expressing, Quantitative RT-PCR, Western Blot, Immunoprecipitation, Immunohistochemistry

Critical roles of TNFR2 in ischemia-induced neovascularization and pericyte recruitment. a: Four weeks after femoral ligation, gastrocnemius muscles were harvested. a: Capillary density and pericyte recruitment were immunostained with CD31 (an EC marker) and SMA (a smooth muscle/pericyte marker). Representative sections from nonischemic and ischemic groups of C57BL/6 mice are shown. b: Representative staining of CD31 and SMA in ischemic groups of TNFR1-KO and TNFR2-KO mice are shown. c–f: Quantification of capillaries (number/mm 2 muscle area), ratio of CD31/muscle fiber, SMA-positive staining (number/mm 2 muscle area), and percentage of SMA-positive staining (SMA/capillaries) are shown. Data from different mice are shown in graphics and n = 4 for each strain. * P
Figure Legend Snippet: Critical roles of TNFR2 in ischemia-induced neovascularization and pericyte recruitment. a: Four weeks after femoral ligation, gastrocnemius muscles were harvested. a: Capillary density and pericyte recruitment were immunostained with CD31 (an EC marker) and SMA (a smooth muscle/pericyte marker). Representative sections from nonischemic and ischemic groups of C57BL/6 mice are shown. b: Representative staining of CD31 and SMA in ischemic groups of TNFR1-KO and TNFR2-KO mice are shown. c–f: Quantification of capillaries (number/mm 2 muscle area), ratio of CD31/muscle fiber, SMA-positive staining (number/mm 2 muscle area), and percentage of SMA-positive staining (SMA/capillaries) are shown. Data from different mice are shown in graphics and n = 4 for each strain. * P

Techniques Used: Ligation, Marker, Mouse Assay, Staining

21) Product Images from "Chikungunya virus impairs draining lymph node function by inhibiting HEV-mediated lymphocyte recruitment"

Article Title: Chikungunya virus impairs draining lymph node function by inhibiting HEV-mediated lymphocyte recruitment

Journal: JCI Insight

doi: 10.1172/jci.insight.121100

Reduced HEV cell expansion in the dLN during pathogenic CHIKV infection. WT C57BL/6 mice were inoculated in both rear footpads with 10 3 PFU of CHIKV 181/25 or AF15561. ( A ) At 5 or 7 dpi, popliteal LNs were collected and analyzed by flow cytometry to identify stromal cell populations. Gating strategy to identify HEVs based on CD45 – CD31 + (top panels) and PNAd + cells (bottom panels). ( B ) Number of CD45 – CD31 + PNAd + HEVs per LN. Data are representative of 3 independent experiments and each bar represents the mean ± SEM of 3–8 mice per group. ( C ) Confocal micrographs showing ERTR-7 + stromal cells (red) and PNAd + HEVs (white) in the dLN at the indicated time points. Images are representative of 3–6 dLNs per group. Scale bars: 200 μm. ( D ) Confocal micrographs showing ER-TR7 + stromal cells (red), B220 + B cells (blue), CD8 + T cells (green), and PNAd + HEVs (white) in the dLN at 5 dpi. Scale bars: 20 μm. ( D ) Total HEV area per section ( n = 3–6 individual mice per group, 1–2 sections analyzed per mouse). ( F ) CCL21 protein levels in the dLN were measured by ELISA at the indicated time points ( n = 5 per group). ( G ) Concentration coefficient for CD8 + fluorescence signal in HEV lumen. * P
Figure Legend Snippet: Reduced HEV cell expansion in the dLN during pathogenic CHIKV infection. WT C57BL/6 mice were inoculated in both rear footpads with 10 3 PFU of CHIKV 181/25 or AF15561. ( A ) At 5 or 7 dpi, popliteal LNs were collected and analyzed by flow cytometry to identify stromal cell populations. Gating strategy to identify HEVs based on CD45 – CD31 + (top panels) and PNAd + cells (bottom panels). ( B ) Number of CD45 – CD31 + PNAd + HEVs per LN. Data are representative of 3 independent experiments and each bar represents the mean ± SEM of 3–8 mice per group. ( C ) Confocal micrographs showing ERTR-7 + stromal cells (red) and PNAd + HEVs (white) in the dLN at the indicated time points. Images are representative of 3–6 dLNs per group. Scale bars: 200 μm. ( D ) Confocal micrographs showing ER-TR7 + stromal cells (red), B220 + B cells (blue), CD8 + T cells (green), and PNAd + HEVs (white) in the dLN at 5 dpi. Scale bars: 20 μm. ( D ) Total HEV area per section ( n = 3–6 individual mice per group, 1–2 sections analyzed per mouse). ( F ) CCL21 protein levels in the dLN were measured by ELISA at the indicated time points ( n = 5 per group). ( G ) Concentration coefficient for CD8 + fluorescence signal in HEV lumen. * P

Techniques Used: Infection, Mouse Assay, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay, Concentration Assay, Fluorescence

Pathogenic, but not attenuated, CHIKV infection impairs the dLN response to footpad NP-OVA immunization. ( A ) WT C57BL/6 mice were mock infected or infected with 181/25 or AF15561. At 7 dpi, mice were left unimmunized (naive) or immunized with 1 g NP 16 -OVA plus alum in the left rear footpad. One or 2 weeks later, the dLN was collected and analyzed for the presence of NP-specific GC B cells by flow cytometry. ( B ) Gating strategy (after gating on single cells) used to identify NP-specific GC B cells. ( C ) Flow plots showing percentage of GC B cells binding to NP-PE. ( D ) Percentage of NP-specific B cells in the dLN at the indicated times after immunization. Data are combined from 2 independent experiments. Each bar represents the mean ± SEM of 4–5 mice per group. * P
Figure Legend Snippet: Pathogenic, but not attenuated, CHIKV infection impairs the dLN response to footpad NP-OVA immunization. ( A ) WT C57BL/6 mice were mock infected or infected with 181/25 or AF15561. At 7 dpi, mice were left unimmunized (naive) or immunized with 1 g NP 16 -OVA plus alum in the left rear footpad. One or 2 weeks later, the dLN was collected and analyzed for the presence of NP-specific GC B cells by flow cytometry. ( B ) Gating strategy (after gating on single cells) used to identify NP-specific GC B cells. ( C ) Flow plots showing percentage of GC B cells binding to NP-PE. ( D ) Percentage of NP-specific B cells in the dLN at the indicated times after immunization. Data are combined from 2 independent experiments. Each bar represents the mean ± SEM of 4–5 mice per group. * P

Techniques Used: Infection, Mouse Assay, Flow Cytometry, Cytometry, Binding Assay

Lymphocyte proliferation in the dLN is similar during acutely cleared and pathogenic CHIKV infection. ( A ) Representative FACS plots and ( B ) number of SIINFEKL tetramer + CD8 + T cells in the spleen of WT C57BL/6 mice ( n = 9 per group) at 7 dpi. ( C – E ) WT C57BL/6 mice ( n = 8 per group) were mock infected or infected with 10 3 PFU 181/25.OVA or AF15561.OVA and the dLN was excised at 5 dpi. ( C ) Frequency of CD8 + SIINFEKL tetramer + cells (top panels) and Ki67 + of total CD8 + cells or tetramer + cells (bottom panels). ( D ) Number of SIINFEKL tetramer + CD8 + T cells. ( E ) Percentage of CD8 + SIINFEKL tetramer + cells expressing Ki67. Data are combined from 2 independent experiments. ( F ) Enriched CD4 + T cells (10 5 ) from CD45.1 + OT-II mice were transferred i.v. into CD45.2 + recipients ( n = 4 per group) 4 hours prior to inoculation with 10 3 PFU 181/25.OVA or AF15561.OVA and the dLN was collected at 4 dpi. ( G ) FACS plots showing Ki67 + OT-II cells. ( H ) Number of transferred OT-II cells per dLN. ( I ) Percentage of Ki67 + OT-II cells. Data are representative of 2 independent experiments. Each bar represents the mean ± SEM. * P
Figure Legend Snippet: Lymphocyte proliferation in the dLN is similar during acutely cleared and pathogenic CHIKV infection. ( A ) Representative FACS plots and ( B ) number of SIINFEKL tetramer + CD8 + T cells in the spleen of WT C57BL/6 mice ( n = 9 per group) at 7 dpi. ( C – E ) WT C57BL/6 mice ( n = 8 per group) were mock infected or infected with 10 3 PFU 181/25.OVA or AF15561.OVA and the dLN was excised at 5 dpi. ( C ) Frequency of CD8 + SIINFEKL tetramer + cells (top panels) and Ki67 + of total CD8 + cells or tetramer + cells (bottom panels). ( D ) Number of SIINFEKL tetramer + CD8 + T cells. ( E ) Percentage of CD8 + SIINFEKL tetramer + cells expressing Ki67. Data are combined from 2 independent experiments. ( F ) Enriched CD4 + T cells (10 5 ) from CD45.1 + OT-II mice were transferred i.v. into CD45.2 + recipients ( n = 4 per group) 4 hours prior to inoculation with 10 3 PFU 181/25.OVA or AF15561.OVA and the dLN was collected at 4 dpi. ( G ) FACS plots showing Ki67 + OT-II cells. ( H ) Number of transferred OT-II cells per dLN. ( I ) Percentage of Ki67 + OT-II cells. Data are representative of 2 independent experiments. Each bar represents the mean ± SEM. * P

Techniques Used: Infection, FACS, Mouse Assay, Expressing

Accumulation of naive lymphocytes in the dLN is impaired during pathogenic CHIKV infection. ( A ) WT C57BL/6 mice ( n = 4 per group) were inoculated with 10 3 PFU of 181/25 or AF15561. At 4.5 dpi, 10 7 naive CFSE-labeled splenocytes were transferred i.v. and enumerated 12 hours later. ( B ) Flow plots depicting the percentage of CFSE-labeled cells in the dLN. ( C ) Total number of CFSE-labeled transferred cells per organ (calculated by multiplying the percentage of CFSE + cells by total cells). Data are representative of 2 independent experiments. Each bar represents the mean ± SEM. * P
Figure Legend Snippet: Accumulation of naive lymphocytes in the dLN is impaired during pathogenic CHIKV infection. ( A ) WT C57BL/6 mice ( n = 4 per group) were inoculated with 10 3 PFU of 181/25 or AF15561. At 4.5 dpi, 10 7 naive CFSE-labeled splenocytes were transferred i.v. and enumerated 12 hours later. ( B ) Flow plots depicting the percentage of CFSE-labeled cells in the dLN. ( C ) Total number of CFSE-labeled transferred cells per organ (calculated by multiplying the percentage of CFSE + cells by total cells). Data are representative of 2 independent experiments. Each bar represents the mean ± SEM. * P

Techniques Used: Infection, Mouse Assay, Labeling, Flow Cytometry

Pathogenic CHIKV infection disrupts the architecture of the draining lymph node. WT C57BL/6 mice were mock infected or infected with 10 3 PFU 181/25 or AF15561 via the left rear footpad and the dLN was excised at the indicated time points (3–8 mice per group). ( A ) H E–stained sections were prepared from paraffin-embedded draining popliteal lymph nodes. Scale bars: 500 μm. Sections were scored for the degree of ( B ) GC development, and ( C ) fibrosis and decreased lymphocytes at the indicated time points. Data represent the mean ± SEM of 3–4 (3 dpi), 5–6 (7 dpi), 7–8 (10 dpi), or 4–5 (14 dpi) mice per group. ** P
Figure Legend Snippet: Pathogenic CHIKV infection disrupts the architecture of the draining lymph node. WT C57BL/6 mice were mock infected or infected with 10 3 PFU 181/25 or AF15561 via the left rear footpad and the dLN was excised at the indicated time points (3–8 mice per group). ( A ) H E–stained sections were prepared from paraffin-embedded draining popliteal lymph nodes. Scale bars: 500 μm. Sections were scored for the degree of ( B ) GC development, and ( C ) fibrosis and decreased lymphocytes at the indicated time points. Data represent the mean ± SEM of 3–4 (3 dpi), 5–6 (7 dpi), 7–8 (10 dpi), or 4–5 (14 dpi) mice per group. ** P

Techniques Used: Infection, Mouse Assay, Staining

22) Product Images from "High-dimensional analysis reveals a pathogenic role of inflammatory monocytes in experimental diffuse alveolar hemorrhage"

Article Title: High-dimensional analysis reveals a pathogenic role of inflammatory monocytes in experimental diffuse alveolar hemorrhage

Journal: JCI Insight

doi: 10.1172/jci.insight.129703

Mass cytometry analysis of lung immune cells in DAH. ( A ) Gross illustration of the spectrum of lung hemorrhage in C57BL/6 mice 2 weeks after pristane injection. ( B ) H E staining of lung tissue from mice treated with pristane or PBS. B, bronchioles; Alv, alveolar space; arrows, hemosiderin deposits. ( C ) viSNE display of mass cytometry comparing isolated lung cells from PBS-treated and pristane-treated mice displayed by cell population identity (concatenated from 5 mice per group with equal sampling). ( D ) Quantification of lung cell populations as percentage of CD45 + cells from mass cytometry analysis ( n = 10 per group). ( E ) viSNE display of mass cytometry analysis by individual lineage identification markers. ( F ) Confocal microscopy of CD11b and Siglec-F expression in isolated lung cells (×100 magnfication). ( G ) Correlation of lung CD11b + myeloid cell expansion or Siglec-F + CD11c + alveolar macrophage depletion with DAH severity score ( n = 10). Data are representative of 3 independent experiments ( A – C , E , and F ) or pooled from 2 independent experiments ( D and G ). Statistical analysis was performed using unpaired Student’s t test ( D ) and Spearman’s correlation ( G ). * P
Figure Legend Snippet: Mass cytometry analysis of lung immune cells in DAH. ( A ) Gross illustration of the spectrum of lung hemorrhage in C57BL/6 mice 2 weeks after pristane injection. ( B ) H E staining of lung tissue from mice treated with pristane or PBS. B, bronchioles; Alv, alveolar space; arrows, hemosiderin deposits. ( C ) viSNE display of mass cytometry comparing isolated lung cells from PBS-treated and pristane-treated mice displayed by cell population identity (concatenated from 5 mice per group with equal sampling). ( D ) Quantification of lung cell populations as percentage of CD45 + cells from mass cytometry analysis ( n = 10 per group). ( E ) viSNE display of mass cytometry analysis by individual lineage identification markers. ( F ) Confocal microscopy of CD11b and Siglec-F expression in isolated lung cells (×100 magnfication). ( G ) Correlation of lung CD11b + myeloid cell expansion or Siglec-F + CD11c + alveolar macrophage depletion with DAH severity score ( n = 10). Data are representative of 3 independent experiments ( A – C , E , and F ) or pooled from 2 independent experiments ( D and G ). Statistical analysis was performed using unpaired Student’s t test ( D ) and Spearman’s correlation ( G ). * P

Techniques Used: Mass Cytometry, Mouse Assay, Injection, Staining, Isolation, Sampling, Confocal Microscopy, Expressing

23) Product Images from "Neutrophil-derived S100 calcium-binding proteins A8/A9 promote reticulated thrombocytosis and atherogenesis in diabetes"

Article Title: Neutrophil-derived S100 calcium-binding proteins A8/A9 promote reticulated thrombocytosis and atherogenesis in diabetes

Journal: The Journal of Clinical Investigation

doi: 10.1172/JCI92450

Interventions to inhibit thrombocytosis in diabetic mice. ( A – F ) SGLT2i studies: C57BL/6 mice were made diabetic with STZ, and blood glucose was lowered with the SGLT2i dapagliflozin (25 mg/kg/d) in the drinking water for 4 weeks. ( A ) Blood glucose levels. ( B and C ) Circulating reticulated platelets ( B ) and Kupffer cell IL-6 expression ( C ) were measured by flow cytometry. ( D ) Plasma TPO levels were quantified by ELISA. ( E and F ) BM progenitor cells ( E ) and megakaryocytes ( F ) were quantified by flow cytometry. n = 6 control and STZ+SGLT2i and n = 4 STZ. * P
Figure Legend Snippet: Interventions to inhibit thrombocytosis in diabetic mice. ( A – F ) SGLT2i studies: C57BL/6 mice were made diabetic with STZ, and blood glucose was lowered with the SGLT2i dapagliflozin (25 mg/kg/d) in the drinking water for 4 weeks. ( A ) Blood glucose levels. ( B and C ) Circulating reticulated platelets ( B ) and Kupffer cell IL-6 expression ( C ) were measured by flow cytometry. ( D ) Plasma TPO levels were quantified by ELISA. ( E and F ) BM progenitor cells ( E ) and megakaryocytes ( F ) were quantified by flow cytometry. n = 6 control and STZ+SGLT2i and n = 4 STZ. * P

Techniques Used: Mouse Assay, Expressing, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay

Reticulated thrombocytosis in diabetes is due to enhanced thrombopoiesis. C57BL/6 mice were made diabetic with STZ and assessed after 4 weeks. ( A – C ) The abundance ( A ) and proliferation ( B ) of BM progenitor cells and STAT5 phosphorylation in MkPs ( C ) were assessed by flow cytometry. ( D ) Bones were sectioned and stained with H E, and megakaryocytes were quantified as cells per field (original magnification, ×20). ( E ) BM megakaryocytes were also quantified by flow cytometry. n = 6 control and n = 8 diabetic. * P
Figure Legend Snippet: Reticulated thrombocytosis in diabetes is due to enhanced thrombopoiesis. C57BL/6 mice were made diabetic with STZ and assessed after 4 weeks. ( A – C ) The abundance ( A ) and proliferation ( B ) of BM progenitor cells and STAT5 phosphorylation in MkPs ( C ) were assessed by flow cytometry. ( D ) Bones were sectioned and stained with H E, and megakaryocytes were quantified as cells per field (original magnification, ×20). ( E ) BM megakaryocytes were also quantified by flow cytometry. n = 6 control and n = 8 diabetic. * P

Techniques Used: Mouse Assay, Flow Cytometry, Cytometry, Staining

Liver phenotype in diabetic mice. ( A – E ) C57BL/6 mice were made diabetic with STZ and assessed after 4 weeks. c-MPL surface expression was measured on BM progenitors ( A ) and circulating platelets ( B ) by flow cytometry. ( C ) Plasma TPO was measured by ELISA. ( D and E ) Abundance of Kupffer cells ( D ) and Kupffer cell IL-6 expression ( E ) were quantified by flow cytometry. n = 6 per group. * P
Figure Legend Snippet: Liver phenotype in diabetic mice. ( A – E ) C57BL/6 mice were made diabetic with STZ and assessed after 4 weeks. c-MPL surface expression was measured on BM progenitors ( A ) and circulating platelets ( B ) by flow cytometry. ( C ) Plasma TPO was measured by ELISA. ( D and E ) Abundance of Kupffer cells ( D ) and Kupffer cell IL-6 expression ( E ) were quantified by flow cytometry. n = 6 per group. * P

Techniques Used: Mouse Assay, Expressing, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay

IL-6 drives reticulated thrombocytosis in diabetes. ( A ) Experimental overview: C57BL/6 WT recipient mice were transplanted with BM from WT or Il6 –/– donor mice, and after 5 weeks a subset of mice were rendered diabetic with STZ. After 4 weeks the mice were analyzed. ( B ) Blood glucose levels. ( C ) Percentage of Kupffer cells in the liver was quantified by flow cytometry. ( D and E ) Percentage ( D ) and numbers ( E ) of circulating reticulated platelets. TO, thiazole orange. ( F and G ) Abundance ( F ) and proliferation ( G ) of MkPs in the BM were quantified by flow cytometry. n = 5 WT groups, n = 6 Il6 –/– controls, and n = 10 Il6 –/– STZ. * P
Figure Legend Snippet: IL-6 drives reticulated thrombocytosis in diabetes. ( A ) Experimental overview: C57BL/6 WT recipient mice were transplanted with BM from WT or Il6 –/– donor mice, and after 5 weeks a subset of mice were rendered diabetic with STZ. After 4 weeks the mice were analyzed. ( B ) Blood glucose levels. ( C ) Percentage of Kupffer cells in the liver was quantified by flow cytometry. ( D and E ) Percentage ( D ) and numbers ( E ) of circulating reticulated platelets. TO, thiazole orange. ( F and G ) Abundance ( F ) and proliferation ( G ) of MkPs in the BM were quantified by flow cytometry. n = 5 WT groups, n = 6 Il6 –/– controls, and n = 10 Il6 –/– STZ. * P

Techniques Used: Mouse Assay, Flow Cytometry, Cytometry

Reticulated thrombocytosis in diabetes. C57BL/6 mice were made diabetic with STZ and assessed after 4 weeks. ( A ) Blood glucose. ( B ) Circulating platelet levels from complete blood counts. ( C and D ) Abundance of circulating reticulated platelets expressed as percentage of platelets of total counts. ( E – I ) Markers of platelet activation: ( E and F ) Reticulated and mature platelet activation was measured by flow cytometry. ( E ) CD62P surface expression. ( F ) Intraplatelet CCL5 levels. ( G ) Plasma CCL5 levels were quantified by ELISA. ( H and I ) Platelet leukocyte aggregates ( H ) and leukocyte activation (CD11b levels) ( I ) were quantified by flow cytometry. n = 6 control and 8 diabetic mice. * P
Figure Legend Snippet: Reticulated thrombocytosis in diabetes. C57BL/6 mice were made diabetic with STZ and assessed after 4 weeks. ( A ) Blood glucose. ( B ) Circulating platelet levels from complete blood counts. ( C and D ) Abundance of circulating reticulated platelets expressed as percentage of platelets of total counts. ( E – I ) Markers of platelet activation: ( E and F ) Reticulated and mature platelet activation was measured by flow cytometry. ( E ) CD62P surface expression. ( F ) Intraplatelet CCL5 levels. ( G ) Plasma CCL5 levels were quantified by ELISA. ( H and I ) Platelet leukocyte aggregates ( H ) and leukocyte activation (CD11b levels) ( I ) were quantified by flow cytometry. n = 6 control and 8 diabetic mice. * P

Techniques Used: Mouse Assay, Activation Assay, Flow Cytometry, Cytometry, Expressing, Enzyme-linked Immunosorbent Assay

24) Product Images from "Utility of a human FcRn transgenic mouse model in drug discovery for early assessment and prediction of human pharmacokinetics of monoclonal antibodies"

Article Title: Utility of a human FcRn transgenic mouse model in drug discovery for early assessment and prediction of human pharmacokinetics of monoclonal antibodies

Journal: mAbs

doi: 10.1080/19420862.2016.1193660

hFcRn Tg Mouse Strain Comparisons. PK profile results of Tg32 hemizygous and homozygous, Tg276 homozygous, WT C57Bl/6 and FcRn KO mice following administration of (A) mAb 14, (B) mAb 04 and (C) mAb17. (D) PK profile overlay in FcRn KO mice for mAb04 and mAb17. (E) PK parameters for mAb14, mAb04 and mAb17 in NHP and each mouse model. Data is depicted as the mean ± standard deviation for 3-6 animals/group for panels A-D.
Figure Legend Snippet: hFcRn Tg Mouse Strain Comparisons. PK profile results of Tg32 hemizygous and homozygous, Tg276 homozygous, WT C57Bl/6 and FcRn KO mice following administration of (A) mAb 14, (B) mAb 04 and (C) mAb17. (D) PK profile overlay in FcRn KO mice for mAb04 and mAb17. (E) PK parameters for mAb14, mAb04 and mAb17 in NHP and each mouse model. Data is depicted as the mean ± standard deviation for 3-6 animals/group for panels A-D.

Techniques Used: Mouse Assay, Standard Deviation

25) Product Images from "IL-25 simultaneously elicits distinct populations of innate lymphoid cells and multipotent progenitor type 2 (MPPtype2) cells"

Article Title: IL-25 simultaneously elicits distinct populations of innate lymphoid cells and multipotent progenitor type 2 (MPPtype2) cells

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20122332

IL-25–elicited MPP type2 cells promote Th2 cytokine–dependent responses in vivo. C57BL/6 WT mice (The Jackson Laboratory) were treated i.p. with 0.3 µg IL-25 daily for 7 d. MLNs were harvested, and MPP type2 cells were sort-purified and injected intradermally into naive C57BL/6 WT mice. (a) IL-4, IL-5, and IL-13 cytokine production from skin-draining LN cells from mice receiving intradermal injection of control or IL-25–elicited MPP type2 cells after 48-h αCD3/αCD28 stimulation measured by ELISA. Data in a are representative of two independent experiments. (b–f) C57BL/6 WT mice were treated i.p. with 0.3 µg IL-25 daily for 7 d. MLNs were harvested, and MPP type2 cells were sort-purified and injected into T. muris –infected (INF) Il17rb −/− mice (Charles River). (b) IFN-γ cytokine production by T. muris antigen–stimulated MLN cells. (c) Total serum IgE antibody titers measured by ELISA. (d) Periodic acid–Schiff/Alcian blue–stained colon sections of intestine tissue from naive or infected WT or Il17rb −/− mice ± MPP type2 cells. N, naive (inset). Bars, 100 µm. (e) Goblet cell counts from d. (f) Worm burdens from T. muris –infected mice were assessed at day 21 after infection. Data in b–f are representative of two independent experiments (WT naive, n = 2–3; WT INF, n = 6–8; Il17rb −/− naive, n = 2–3; Il17rb −/− INF, n = 6–7; Il17rb −/− INF + MPP type2 cells, n = 6). *, P
Figure Legend Snippet: IL-25–elicited MPP type2 cells promote Th2 cytokine–dependent responses in vivo. C57BL/6 WT mice (The Jackson Laboratory) were treated i.p. with 0.3 µg IL-25 daily for 7 d. MLNs were harvested, and MPP type2 cells were sort-purified and injected intradermally into naive C57BL/6 WT mice. (a) IL-4, IL-5, and IL-13 cytokine production from skin-draining LN cells from mice receiving intradermal injection of control or IL-25–elicited MPP type2 cells after 48-h αCD3/αCD28 stimulation measured by ELISA. Data in a are representative of two independent experiments. (b–f) C57BL/6 WT mice were treated i.p. with 0.3 µg IL-25 daily for 7 d. MLNs were harvested, and MPP type2 cells were sort-purified and injected into T. muris –infected (INF) Il17rb −/− mice (Charles River). (b) IFN-γ cytokine production by T. muris antigen–stimulated MLN cells. (c) Total serum IgE antibody titers measured by ELISA. (d) Periodic acid–Schiff/Alcian blue–stained colon sections of intestine tissue from naive or infected WT or Il17rb −/− mice ± MPP type2 cells. N, naive (inset). Bars, 100 µm. (e) Goblet cell counts from d. (f) Worm burdens from T. muris –infected mice were assessed at day 21 after infection. Data in b–f are representative of two independent experiments (WT naive, n = 2–3; WT INF, n = 6–8; Il17rb −/− naive, n = 2–3; Il17rb −/− INF, n = 6–7; Il17rb −/− INF + MPP type2 cells, n = 6). *, P

Techniques Used: In Vivo, Mouse Assay, Purification, Injection, Enzyme-linked Immunosorbent Assay, Infection, Staining

IL-25 elicits ILC2 and MPP type2 cells independent of IL-33 signaling. (a–d) C57BL/6 WT mice and C57BL/6 Il33 −/− mice (Taconic) were treated i.p. with PBS (control) or 0.3 µg of recombinant IL-25 daily for 7 d. MLNs were harvested at day 7, and the frequency and total cell numbers of ILC2 and MPP type2 cells were assessed by flow cytometry. (a and c) Frequency of ILC2 in control or IL-25–treated WT (a) or Il33 −/− mice (c). (b and d) Total cell numbers of ILC2 in control or IL-25–treated WT (b) or Il33 −/− mice (d). (e and g) Frequency of MPP type2 cells in control or IL-25–treated WT (e) or Il33 −/− mice (g). (f and h) Total cell numbers of MPP type2 cells in control or IL-25–treated WT (f) or Il33 −/− mice (h). Data in a–h are representative of two independent experiments (control, n = 4; IL-25–treated WT, n = 6; IL-25–treated Il33 −/− , n = 6). (i–l) BALB/c WT mice (The Jackson Laboratory) were treated with PBS (control) or 0.3 µg of recombinant IL-25 plus isotype (IgG) or anti-T1/ST2 mAbs. MLNs were harvested at day 7, and the frequency and total cell numbers of ILC2 and MPP type2 cells were assessed by flow cytometry. Frequency and total numbers of T1/ST2 pos IL-7Rα pos ILC2 (i and j) and T1/ST2 neg IL-7Rα neg c-kit pos MPP type2 cells (k and l). Plots are gated on live, Lin neg (CD4, CD8α, CD11b, CD11c, and CD19) cells. Data in i–l are representative of two independent experiments (control, n = 6; IL-25 treated, n = 9; anti-T1/ST2 IL-25 treated, n = 9). Error bars indicate SEM.
Figure Legend Snippet: IL-25 elicits ILC2 and MPP type2 cells independent of IL-33 signaling. (a–d) C57BL/6 WT mice and C57BL/6 Il33 −/− mice (Taconic) were treated i.p. with PBS (control) or 0.3 µg of recombinant IL-25 daily for 7 d. MLNs were harvested at day 7, and the frequency and total cell numbers of ILC2 and MPP type2 cells were assessed by flow cytometry. (a and c) Frequency of ILC2 in control or IL-25–treated WT (a) or Il33 −/− mice (c). (b and d) Total cell numbers of ILC2 in control or IL-25–treated WT (b) or Il33 −/− mice (d). (e and g) Frequency of MPP type2 cells in control or IL-25–treated WT (e) or Il33 −/− mice (g). (f and h) Total cell numbers of MPP type2 cells in control or IL-25–treated WT (f) or Il33 −/− mice (h). Data in a–h are representative of two independent experiments (control, n = 4; IL-25–treated WT, n = 6; IL-25–treated Il33 −/− , n = 6). (i–l) BALB/c WT mice (The Jackson Laboratory) were treated with PBS (control) or 0.3 µg of recombinant IL-25 plus isotype (IgG) or anti-T1/ST2 mAbs. MLNs were harvested at day 7, and the frequency and total cell numbers of ILC2 and MPP type2 cells were assessed by flow cytometry. Frequency and total numbers of T1/ST2 pos IL-7Rα pos ILC2 (i and j) and T1/ST2 neg IL-7Rα neg c-kit pos MPP type2 cells (k and l). Plots are gated on live, Lin neg (CD4, CD8α, CD11b, CD11c, and CD19) cells. Data in i–l are representative of two independent experiments (control, n = 6; IL-25 treated, n = 9; anti-T1/ST2 IL-25 treated, n = 9). Error bars indicate SEM.

Techniques Used: Mouse Assay, Recombinant, Flow Cytometry, Cytometry

IL-25–mediated induction of MPP type2 cells and type 2 inflammation occurs independently of ILC2. (a–i) C57BL/6 Rag1 −/− mice (The Jackson Laboratory) were treated i.p. with PBS (control) or 0.3 µg of IL-25 plus isotype mAb (Iso.) or anti-CD90 mAb. (a and b) Frequencies (a) and total cell numbers (b) of CD90 pos T1/ST2 pos IL-7Rα pos ILC2 in the MLNs of control or IL-25–treated mice given either isotype or anti-CD90 mAb. (c and d) Frequencies (c) and total cell numbers (d) of CD90 neg T1/ST2 neg IL-7Rα neg MPP type2 cells in the MLNs of control or IL-25–treated mice given either isotype or anti-CD90 mAb. Plots are gated on live, Lin neg (CD4, CD8, CD11b, CD11c, and CD19) cells or as indicated. (e and f) Quantitative real-time PCR of Il4 , Il5 , and Il13 gene expression levels from lung (e) or small intestinal tissue (f) of control or IL-25–treated mice receiving either isotype or anti-CD90 mAb. RQ, relative quantification. (g) Periodic acid–Schiff/Alcian blue staining of lung and small intestine (SI) tissue from control or IL-25–treated mice receiving either isotype or anti-CD90 mAb. Bars: (top) 50 µm; (bottom) 100 µm. (h) Goblet cell (GC) counts from g. nd, not detected. (i) Frequency and total cell numbers of eosinophils in the lung from control or IL-25–treated mice given either isotype or anti-CD90 mAb. Frequency is percentage of SSC hi SiglecF pos cells gated on live, Lin neg CD11b pos cells. Data in a–i are representative of three independent experiments (control + Iso., n = 6; control + anti-CD90, n = 6; IL-25 treated + Iso., n = 6; IL-25 treated + anti-CD90, n = 6). *, P
Figure Legend Snippet: IL-25–mediated induction of MPP type2 cells and type 2 inflammation occurs independently of ILC2. (a–i) C57BL/6 Rag1 −/− mice (The Jackson Laboratory) were treated i.p. with PBS (control) or 0.3 µg of IL-25 plus isotype mAb (Iso.) or anti-CD90 mAb. (a and b) Frequencies (a) and total cell numbers (b) of CD90 pos T1/ST2 pos IL-7Rα pos ILC2 in the MLNs of control or IL-25–treated mice given either isotype or anti-CD90 mAb. (c and d) Frequencies (c) and total cell numbers (d) of CD90 neg T1/ST2 neg IL-7Rα neg MPP type2 cells in the MLNs of control or IL-25–treated mice given either isotype or anti-CD90 mAb. Plots are gated on live, Lin neg (CD4, CD8, CD11b, CD11c, and CD19) cells or as indicated. (e and f) Quantitative real-time PCR of Il4 , Il5 , and Il13 gene expression levels from lung (e) or small intestinal tissue (f) of control or IL-25–treated mice receiving either isotype or anti-CD90 mAb. RQ, relative quantification. (g) Periodic acid–Schiff/Alcian blue staining of lung and small intestine (SI) tissue from control or IL-25–treated mice receiving either isotype or anti-CD90 mAb. Bars: (top) 50 µm; (bottom) 100 µm. (h) Goblet cell (GC) counts from g. nd, not detected. (i) Frequency and total cell numbers of eosinophils in the lung from control or IL-25–treated mice given either isotype or anti-CD90 mAb. Frequency is percentage of SSC hi SiglecF pos cells gated on live, Lin neg CD11b pos cells. Data in a–i are representative of three independent experiments (control + Iso., n = 6; control + anti-CD90, n = 6; IL-25 treated + Iso., n = 6; IL-25 treated + anti-CD90, n = 6). *, P

Techniques Used: Mouse Assay, Real-time Polymerase Chain Reaction, Expressing, Staining

IL-25–elicited MPP type2 cells possess a unique transcriptional profile from ILC2 cells. (a and b) C57BL/6 WT mice (The Jackson Laboratory) were treated with 0.3 µg of recombinant IL-25 daily for 7 d. MLNs and PECs were harvested from IL-25–treated mice and MPP type2 cells (Lin neg T1/ST2 neg IL-7Rα neg CD4 neg CD90 neg CD25 neg c-kit pos ; a) were FACS purified to ≥95% purity (b). Three biological replicates of MPP type2 cells were collected, and mRNA was isolated, amplified, and hybridized to Affymetrix gene chips for microarray analysis. The previously published microarray gene expression profile of lung-resident ILC2 was used for comparison (GEO series no. GSE46468 ; Monticelli et al., 2011 ). (c) Heat map representing gene expression profiles of the top 100 differentially expressed genes between MPP type2 cells and ILC2. Red indicates high expression, and blue indicates low expression. (d) GSEA comparing the gene expression signatures of MPP type2 cells and ILC2. (e) List of leading edge genes from GSEA analysis from d. (f) PCA plot comparing transcriptional profiles for MPP type2 cells (1), ex vivo nuocytes (2; GSE25890 ), NHCs (3; GSE18752 ), lung-resident ILC2 (4; GSE46468 ), unstimulated lung NHCs (5; GSE36057 ), splenic LTi cells (6, GSE46468 ; and 7, GSE18752 ), and BM-GMPs (8). Categories of ILC2 (green shaded area), ILC3 (red shaded area), and progenitors (blue shaded area) were grouped (as indicated in f, dashed lines), and Euclidean distance measurements between MPP type2 cells and BM-GMP versus ILC2 or ILC3 were calculated (g). ***, P
Figure Legend Snippet: IL-25–elicited MPP type2 cells possess a unique transcriptional profile from ILC2 cells. (a and b) C57BL/6 WT mice (The Jackson Laboratory) were treated with 0.3 µg of recombinant IL-25 daily for 7 d. MLNs and PECs were harvested from IL-25–treated mice and MPP type2 cells (Lin neg T1/ST2 neg IL-7Rα neg CD4 neg CD90 neg CD25 neg c-kit pos ; a) were FACS purified to ≥95% purity (b). Three biological replicates of MPP type2 cells were collected, and mRNA was isolated, amplified, and hybridized to Affymetrix gene chips for microarray analysis. The previously published microarray gene expression profile of lung-resident ILC2 was used for comparison (GEO series no. GSE46468 ; Monticelli et al., 2011 ). (c) Heat map representing gene expression profiles of the top 100 differentially expressed genes between MPP type2 cells and ILC2. Red indicates high expression, and blue indicates low expression. (d) GSEA comparing the gene expression signatures of MPP type2 cells and ILC2. (e) List of leading edge genes from GSEA analysis from d. (f) PCA plot comparing transcriptional profiles for MPP type2 cells (1), ex vivo nuocytes (2; GSE25890 ), NHCs (3; GSE18752 ), lung-resident ILC2 (4; GSE46468 ), unstimulated lung NHCs (5; GSE36057 ), splenic LTi cells (6, GSE46468 ; and 7, GSE18752 ), and BM-GMPs (8). Categories of ILC2 (green shaded area), ILC3 (red shaded area), and progenitors (blue shaded area) were grouped (as indicated in f, dashed lines), and Euclidean distance measurements between MPP type2 cells and BM-GMP versus ILC2 or ILC3 were calculated (g). ***, P

Techniques Used: Mouse Assay, Recombinant, FACS, Purification, Isolation, Amplification, Microarray, Expressing, Ex Vivo

IL-25 simultaneously elicits phenotypically distinct populations of MPP type2 cells and ILC2. (a–r) C57BL/6 WT mice (The Jackson Laboratory) were treated i.p. with PBS (control), 0.3 µg of recombinant IL-33, or 0.3 µg of recombinant IL-25 daily for 2, 4, or 7 d. Indicated tissues were harvested at day 7 (or as indicated), and the frequency and total cell numbers of ILC2 and MPP type2 cells were assessed by flow cytometry. (a) Frequency of T1/ST2 pos IL-7Rα pos ILC2 in the Lin neg cell compartment from the MLNs of control or IL-33–treated mice. (b) CD90.2 (Thy1.2) and CD25 expression on ILC2 (black histograms) from IL-33–treated mice. (c) Total cell numbers of ILC2 from control or IL-33–treated mice. (d) Expression of c-kit on gated T1/ST2 neg IL-7Rα neg cells from control or IL-33–treated mice. (e) CD90 and CD25 expression on c-kit pos MPP type2 cells (black histograms) from IL-33–treated mice. (f) Total cell numbers of MPP type2 cells from control or IL-33–treated mice. (g) Frequency of ILC2 in the Lin neg cell compartment from the MLN of control or IL-25–treated mice. (h) CD90 and CD25 expression on ILC2 (black histograms) from IL-25–treated mice. (i) Total cell numbers of ILC2 from control or IL-25–treated mice. (j) Expression of c-kit on gated T1/ST2 neg IL-7Rα neg cells from control or IL-25–treated mice. (k) CD90 and CD25 expression on c-kit pos MPP type2 cells (black histograms) from IL-25–treated mice. (l) Total cell numbers of MPP type2 cells from control or IL-25–treated mice. Plots are gated on live, Lin neg (CD4, CD8, CD11b, CD11c, and CD19) cells or as indicated. Gray shaded histograms represent CD90 or CD25 expression on Lin neg cells from control mice. (m and n) Frequencies of ILC2 (m) or MPP type2 cells (n) as gated in a, g, d, and i from the MLNs of control (open bars), IL-33–treated, or IL-25–treated mice at days 2, 4, and 6. (o–r) Frequencies of ILC2 and MPP type2 cells as gated in a and g and d and i from the blood (o), caudal LN (p), lung (q), and PEC (r) of control (open bars), IL-33–treated (black bars), or IL-25–treated (gray bars) mice at day 6. Data in a–n are representative of two or more independent experiments (control, n = 4; IL-33 treated, n = 8; IL-25 treated, n = 8). Data in o–r are representative of two independent experiments (control, n = 4; IL-25 treated, n = 8; IL-33 treated, n = 8). *, P
Figure Legend Snippet: IL-25 simultaneously elicits phenotypically distinct populations of MPP type2 cells and ILC2. (a–r) C57BL/6 WT mice (The Jackson Laboratory) were treated i.p. with PBS (control), 0.3 µg of recombinant IL-33, or 0.3 µg of recombinant IL-25 daily for 2, 4, or 7 d. Indicated tissues were harvested at day 7 (or as indicated), and the frequency and total cell numbers of ILC2 and MPP type2 cells were assessed by flow cytometry. (a) Frequency of T1/ST2 pos IL-7Rα pos ILC2 in the Lin neg cell compartment from the MLNs of control or IL-33–treated mice. (b) CD90.2 (Thy1.2) and CD25 expression on ILC2 (black histograms) from IL-33–treated mice. (c) Total cell numbers of ILC2 from control or IL-33–treated mice. (d) Expression of c-kit on gated T1/ST2 neg IL-7Rα neg cells from control or IL-33–treated mice. (e) CD90 and CD25 expression on c-kit pos MPP type2 cells (black histograms) from IL-33–treated mice. (f) Total cell numbers of MPP type2 cells from control or IL-33–treated mice. (g) Frequency of ILC2 in the Lin neg cell compartment from the MLN of control or IL-25–treated mice. (h) CD90 and CD25 expression on ILC2 (black histograms) from IL-25–treated mice. (i) Total cell numbers of ILC2 from control or IL-25–treated mice. (j) Expression of c-kit on gated T1/ST2 neg IL-7Rα neg cells from control or IL-25–treated mice. (k) CD90 and CD25 expression on c-kit pos MPP type2 cells (black histograms) from IL-25–treated mice. (l) Total cell numbers of MPP type2 cells from control or IL-25–treated mice. Plots are gated on live, Lin neg (CD4, CD8, CD11b, CD11c, and CD19) cells or as indicated. Gray shaded histograms represent CD90 or CD25 expression on Lin neg cells from control mice. (m and n) Frequencies of ILC2 (m) or MPP type2 cells (n) as gated in a, g, d, and i from the MLNs of control (open bars), IL-33–treated, or IL-25–treated mice at days 2, 4, and 6. (o–r) Frequencies of ILC2 and MPP type2 cells as gated in a and g and d and i from the blood (o), caudal LN (p), lung (q), and PEC (r) of control (open bars), IL-33–treated (black bars), or IL-25–treated (gray bars) mice at day 6. Data in a–n are representative of two or more independent experiments (control, n = 4; IL-33 treated, n = 8; IL-25 treated, n = 8). Data in o–r are representative of two independent experiments (control, n = 4; IL-25 treated, n = 8; IL-33 treated, n = 8). *, P

Techniques Used: Mouse Assay, Recombinant, Flow Cytometry, Cytometry, Expressing

IL-25–mediated induction of MPP type2 cells occurs independently of Id2. (a–p) CD5/B220-depleted donor BM (WT, CD45.1; Id2 −/− , CD45.1.2) was transferred into lethally irradiated C57BL/6 WT (CD45.2) mice. After reconstitution for 8 wk after transplant, mice were treated i.p. with PBS (control), 0.3 µg of recombinant IL-33, or 0.3 µg of recombinant IL-25 daily for 7 d. MLNs were harvested, and the frequency and total cell numbers of ILC2 and MPP type2 cells were assessed by flow cytometry. (a and c) Frequency of T1/ST2 pos IL-7Rα pos ILC2 from control or IL-33–treated WT BM chimera mice (a) or Id2-deficient BM chimera mice (c). (b and d) Total cell numbers of ILC2 from control or IL-33–treated WT BM chimera mice (b) or Id2-deficient BM chimera mice (d). (e and g) Frequency of c-kit pos MPP type2 cells from control or IL-33–treated WT BM chimera mice (e) or Id2-deficient BM chimera mice (g). (f and h) Total cell numbers of MPP type2 cells from control or IL-33–treated WT BM chimera mice (f) or Id2-deficient BM chimera mice (h). (i and k) Frequency of T1/ST2 pos IL-7Rα pos ILC2 cells from control or IL-25–treated WT BM chimera mice (i) or Id2-deficient BM chimera mice (k). (j and l) Total cell numbers of ILC2 from control or IL-25–treated WT BM chimera mice (j) or Id2-deficient BM chimera mice (l). (m and o) Frequency of c-kit pos MPP type2 cells from control or IL-25–treated WT BM chimera mice (m) or Id2-deficient BM chimera mice (o). (n and p) Total cell numbers of MPP type2 cells from control or IL-25–treated WT BM chimera mice (n) or Id2-deficient BM chimera mice (p). Plots are gated on live, Lin neg (CD4, CD8, CD11b, CD11c, and CD19) donor-derived cells. Data in a–p are representative of two or more independent experiments (control, n = 4; IL-33 treated, n = 6; IL-25 treated, n = 6). Error bars indicate SEM.
Figure Legend Snippet: IL-25–mediated induction of MPP type2 cells occurs independently of Id2. (a–p) CD5/B220-depleted donor BM (WT, CD45.1; Id2 −/− , CD45.1.2) was transferred into lethally irradiated C57BL/6 WT (CD45.2) mice. After reconstitution for 8 wk after transplant, mice were treated i.p. with PBS (control), 0.3 µg of recombinant IL-33, or 0.3 µg of recombinant IL-25 daily for 7 d. MLNs were harvested, and the frequency and total cell numbers of ILC2 and MPP type2 cells were assessed by flow cytometry. (a and c) Frequency of T1/ST2 pos IL-7Rα pos ILC2 from control or IL-33–treated WT BM chimera mice (a) or Id2-deficient BM chimera mice (c). (b and d) Total cell numbers of ILC2 from control or IL-33–treated WT BM chimera mice (b) or Id2-deficient BM chimera mice (d). (e and g) Frequency of c-kit pos MPP type2 cells from control or IL-33–treated WT BM chimera mice (e) or Id2-deficient BM chimera mice (g). (f and h) Total cell numbers of MPP type2 cells from control or IL-33–treated WT BM chimera mice (f) or Id2-deficient BM chimera mice (h). (i and k) Frequency of T1/ST2 pos IL-7Rα pos ILC2 cells from control or IL-25–treated WT BM chimera mice (i) or Id2-deficient BM chimera mice (k). (j and l) Total cell numbers of ILC2 from control or IL-25–treated WT BM chimera mice (j) or Id2-deficient BM chimera mice (l). (m and o) Frequency of c-kit pos MPP type2 cells from control or IL-25–treated WT BM chimera mice (m) or Id2-deficient BM chimera mice (o). (n and p) Total cell numbers of MPP type2 cells from control or IL-25–treated WT BM chimera mice (n) or Id2-deficient BM chimera mice (p). Plots are gated on live, Lin neg (CD4, CD8, CD11b, CD11c, and CD19) donor-derived cells. Data in a–p are representative of two or more independent experiments (control, n = 4; IL-33 treated, n = 6; IL-25 treated, n = 6). Error bars indicate SEM.

Techniques Used: Irradiation, Mouse Assay, Recombinant, Flow Cytometry, Cytometry, Derivative Assay

26) Product Images from "CXCR2- and E-Selectin-induced Neutrophil Arrest during Inflammation In Vivo"

Article Title: CXCR2- and E-Selectin-induced Neutrophil Arrest during Inflammation In Vivo

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20040424

Numbers of adherent cells per mm 2 in murine cremaster muscle venules. Cremaster muscle exteriorized 3 h after intrascrotal injection of 500 ng TNF-α in WT (11 venules in 3 mice), WT plus 4 μg PTx 5 min before TNF-α injection (12 venules in 3 mice), E-selectin −/− (13 venules in 3 mice), E-selectin −/− plus PTx (17 venules in 3 mice), and WT plus PTx plus 60 μg α–E-selectin mAb 9A9 5 min before TNF-α injection (10 venules in 2 mice) using mice on a C57Bl/6 background (A), and WT (11 venules in 2 mice), CXCR2 −/− (13 venules in 3 mice), and CXCR2 −/− plus mAb 9A9 (11 venules in 2 mice) using mice on a BALB/c background (B). *, P
Figure Legend Snippet: Numbers of adherent cells per mm 2 in murine cremaster muscle venules. Cremaster muscle exteriorized 3 h after intrascrotal injection of 500 ng TNF-α in WT (11 venules in 3 mice), WT plus 4 μg PTx 5 min before TNF-α injection (12 venules in 3 mice), E-selectin −/− (13 venules in 3 mice), E-selectin −/− plus PTx (17 venules in 3 mice), and WT plus PTx plus 60 μg α–E-selectin mAb 9A9 5 min before TNF-α injection (10 venules in 2 mice) using mice on a C57Bl/6 background (A), and WT (11 venules in 2 mice), CXCR2 −/− (13 venules in 3 mice), and CXCR2 −/− plus mAb 9A9 (11 venules in 2 mice) using mice on a BALB/c background (B). *, P

Techniques Used: Injection, Mouse Assay

Related Articles

Knock-Out:

Article Title: Respiratory Syncytial Virus (RSV) Infection in Elderly Mice Results in Altered Antiviral Gene Expression and Enhanced Pathology
Article Snippet: .. OPN-/- or knockout (OPN KO) were purchased from Jackson Laboratory (strain B6.Cg-Spp1tm1Blh/J) and were backcrossed with wildtype (WT) C57BL/6 that were also purchased from Jackson Laboratories. ..

Mouse Assay:

Article Title: Regulation of murine NK cell exhaustion through the activation of the DNA damage repair pathway
Article Snippet: .. WT C57BL/6 (H-2b ) and BALB/c (H-2d ) mice were purchased from The Jackson Laboratory. .. C57BL/6 KLRK1–/– (NKG2D–/– ) mice were a gift from David Raulet (University of California, Berkeley, Berkeley, California, USA) and bred in our animal facility.

Article Title: CD8? Dendritic Cells Drive Establishment of HSV-1 Latency
Article Snippet: .. WT C57BL/6, C57BL/6-CD8α−/− , C57BL/6-β2 m−/− , C57BL/6-DTR, C57BL/6-GFP, BXH2/TyJ and C3H/HEJ mice were purchased from Jackson Laboratories. .. C57BL/6-CD8β−/− mice have been reported previously and were bred in-house.

Article Title: Autocrine Effects of Tumor-Derived Complement
Article Snippet: .. Female athymic NU/NU mice were purchased from Taconic and WT C57BL/6, C3 −/− , and CD8 −/− mice from The Jackson Laboratory. .. Mice were cared for in accordance with guidelines set forth by the American Association for Accreditation of Laboratory Animal Care and the US Public Health Service Policy on Human Care and Use of Laboratory Animals.

Article Title: Toll-Like Receptor 4-Dependent Early Elicited Tumor Necrosis Factor Alpha Expression Is Critical for Innate Host Defense against Bordetella bronchiseptica
Article Snippet: .. WT C57BL/6 and C57BL/10ScSn mice, TLR4-deficient C3H/HeJ and C57BL/10ScNCr mice, and TNF-α−/− mice were obtained from Jackson Laboratories. .. WT C3H/HeN mice were obtained from Charles River Laboratories.

Article Title: IL-1RA regulates immunopathogenesis during fungal-associated allergic airway inflammation
Article Snippet: .. WT C57BL/6, Il1r1 –/– (IL-1R1), and Il1rn –/– (IL-1RA) male mice, 6–8 weeks of age, were obtained from The Jackson Laboratory. .. A . fumigatus isolate 13073 (ATCC) was maintained on potato dextrose agar for 5–7 days at 37°C.

Article Title: Blockade of self-reactive IgM significantly reduces injury in a murine model of acute myocardial infarction
Article Snippet: .. Both WT C57BL/6 and RAG-1−/− mice on a C57BL/6 genetic background were purchased from The Jackson Laboratory and housed under specific pathogen-free conditions at the Immune Disease Institute. ..

Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 91
    The Jackson Laboratory tcrδ knockout mice wild type wt c57bl 6
    IgA and IgG2b levels are elevated in <t>TCRδ</t> −/− mice. Levels of Ig isotypes were determined by ELISA in serum (A), fecal pellet (B), and peritoneal lavage (C) from WT <t>(C57BL/6)</t> and TCRδ −/− mice. Data are expressed as means±standard error of the mean (n=3 mice). n.s., not significant. * p
    Tcrδ Knockout Mice Wild Type Wt C57bl 6, supplied by The Jackson Laboratory, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/tcrδ knockout mice wild type wt c57bl 6/product/The Jackson Laboratory
    Average 91 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    tcrδ knockout mice wild type wt c57bl 6 - by Bioz Stars, 2021-01
    91/100 stars
      Buy from Supplier

    88
    The Jackson Laboratory c57bl 6 wt control mice
    Impaired IgG and GC responses in CD154 TG CD22 −/− mice. ( A ) Serum IgM and IgG levels of 4 and 12 mo-old WT, CD22 −/− , CD154 TG , and CD154 TG CD22 −/− mice. Symbols represent serum concentrations for individual mice as determined by ELISA, with means indicated by horizontal bars. ( B ) Serum autoAbs reactive with dsDNA, ssDNA, or histone proteins in 12 mo-old mice. ELISA OD values for IgM (upper panels) and IgG (lower panels) autoAbs are shown for individual mice, with means indicated by horizontal bars. Sera from 2 mo-old WT <t>C57BL/6</t> and 6 mo-old MLR lpr mice were used as negative and positive controls, respectively. ( C ) Impaired IgG responses to a TD Ag. WT ( n = 3), CD22 −/− ( n = 4), CD154 TG ( n = 6), and CD154 TG CD22 −/− ( n = 8) mice were immunized with DNP-KLH in adjuvant on day 0, and boosted on day 21. The graph shows mean (±SEM) DNP-specific IgG levels as determined by ELISA. Images on the right represent immunofluorescence staining of frozen spleen sections from all genotypes harvested 7 days after the boost phase of DNP-KLH immunization. Merged images show the presence of B220 + B cells (red) and GC GL7 + B220 + B cells (yellow). Enlarged regions from these sections indicate typical GC structures present within the follicles of WT and CD22 −/− mice, and detectable GL7 + B220 + B cells within the follicles of CD154 TG mice, but not in CD154 TG CD22 −/− mice (representative regions are shown for comparison). (A–C) Means significantly different from WT are indicated by asterisks (*p≤0.05, **p≤0.01), and between other indicated groups by crosses (†p
    C57bl 6 Wt Control Mice, supplied by The Jackson Laboratory, used in various techniques. Bioz Stars score: 88/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/c57bl 6 wt control mice/product/The Jackson Laboratory
    Average 88 stars, based on 7 article reviews
    Price from $9.99 to $1999.99
    c57bl 6 wt control mice - by Bioz Stars, 2021-01
    88/100 stars
      Buy from Supplier

    91
    The Jackson Laboratory strain matched wt c57bl 6 mice
    LTB 4 is required for IL-1β production. A) Quantification of IL-1β and B) Il1b mRNA expression in the skin of <t>C57BL/6,</t> Ltb4r1 −/− , C57BL/6 treated with a topical ointment containing LTB 4 or C57BL/6 treated with a topical ointment containing BLT1 antagonist U-75302 24h after MRSA infection. C) IVIS scanning of pIL1DsRed mice treated with a topical ointment containing BLT1 antagonist U-75032 or vehicle. TNF-α quantification in D) peritoneal macrophages treated with LTB 4 or BLT1 antagonist before MRSA infection and E) skin biopsy from wild-type (WT) or Ltb4r1 −/− mice after 24h MRSA infection. F) IL-1β quantification from human neutrophils isolated from healthy blood donors treated with LTB 4 and LPS. Data are the mean ± SEM of 3-10 mice. *p
    Strain Matched Wt C57bl 6 Mice, supplied by The Jackson Laboratory, used in various techniques. Bioz Stars score: 91/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/strain matched wt c57bl 6 mice/product/The Jackson Laboratory
    Average 91 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    strain matched wt c57bl 6 mice - by Bioz Stars, 2021-01
    91/100 stars
      Buy from Supplier

    88
    The Jackson Laboratory wt c57bl 6 male mice
    Protective efficacy of anti-JEV MAbs in mice. (A and B) Four- to 5-week-old male <t>C57BL/6</t> mice were passively administered 10 µg of the indicated (A) mouse or (B) human MAb via intraperitoneal injection 1 day prior to inoculation with 10 2 FFU of JEV-Nakayama via the subcutaneous route. JEV-31 ( n = 9), JEV-106 ( n = 8), JEV-143 ( n = 8), and JEV-169 ( n = 10) provided complete protection against lethality. JEV-27 ( n = 8), JEV-128 ( n = 9), and JEV-131 ( n = 9) provided partial protection compared to the isotype control MAbs. (C and D) Three-week-old male C57BL/6 mice were passively administered 10 µg of the indicated MAb as described above 1 day prior to inoculation with 10 3 FFU of (C) JEV-MAR 859 (JEV-31, n = 8; JEV-131, n = 9; JEV-169, n = 8) or (D) JEV-2372/79 (JEV-31, n = 9; JEV-106, n = 9; JEV-131, n = 9; JEV-169, n = 9). (E and F) Two hundred fifty micrograms of the indicated MAb was administered 5 days postinfection to (E) 4- to 5-week-old mice infected with 10 2 FFU of JEV-Nakayama (JEV-31, n = 9; JEV-106, n = 9; JEV-143, n = 9; JEV-169, n = 9; hJEV-75, n = 8) or (F) 3-week-old mice infected with 10 3 FFU of JEV-2372/79 (JEV-31, n = 10; JEV-131, n = 9; JEV-143, n = 9; JEV-169, n = 10; hJEV-75, n = 9). Data are pooled from at least two independent experiments. Survival was analyzed for each MAb compared to the isotype control MAb by the log rank test. *, P
    Wt C57bl 6 Male Mice, supplied by The Jackson Laboratory, used in various techniques. Bioz Stars score: 88/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/wt c57bl 6 male mice/product/The Jackson Laboratory
    Average 88 stars, based on 11 article reviews
    Price from $9.99 to $1999.99
    wt c57bl 6 male mice - by Bioz Stars, 2021-01
    88/100 stars
      Buy from Supplier

    Image Search Results


    IgA and IgG2b levels are elevated in TCRδ −/− mice. Levels of Ig isotypes were determined by ELISA in serum (A), fecal pellet (B), and peritoneal lavage (C) from WT (C57BL/6) and TCRδ −/− mice. Data are expressed as means±standard error of the mean (n=3 mice). n.s., not significant. * p

    Journal: Immune Network

    Article Title: Murine γδ T Cells Render B Cells Refractory to Commitment of IgA Isotype Switching

    doi: 10.4110/in.2018.18.e25

    Figure Lengend Snippet: IgA and IgG2b levels are elevated in TCRδ −/− mice. Levels of Ig isotypes were determined by ELISA in serum (A), fecal pellet (B), and peritoneal lavage (C) from WT (C57BL/6) and TCRδ −/− mice. Data are expressed as means±standard error of the mean (n=3 mice). n.s., not significant. * p

    Article Snippet: Animals and genotyping of TCRδ knockout mice Wild-type (WT) C57BL/6 (Daehan Biolink, Seoul, Korea) and TCRδ−/− mice ( ) (JAX stock #002119, Jackson Lab) were maintained on an 8:16-h light:dark cycle in an animal environmental control chamber (Daehan Biolink).

    Techniques: Mouse Assay, Enzyme-linked Immunosorbent Assay

    Impaired IgG and GC responses in CD154 TG CD22 −/− mice. ( A ) Serum IgM and IgG levels of 4 and 12 mo-old WT, CD22 −/− , CD154 TG , and CD154 TG CD22 −/− mice. Symbols represent serum concentrations for individual mice as determined by ELISA, with means indicated by horizontal bars. ( B ) Serum autoAbs reactive with dsDNA, ssDNA, or histone proteins in 12 mo-old mice. ELISA OD values for IgM (upper panels) and IgG (lower panels) autoAbs are shown for individual mice, with means indicated by horizontal bars. Sera from 2 mo-old WT C57BL/6 and 6 mo-old MLR lpr mice were used as negative and positive controls, respectively. ( C ) Impaired IgG responses to a TD Ag. WT ( n = 3), CD22 −/− ( n = 4), CD154 TG ( n = 6), and CD154 TG CD22 −/− ( n = 8) mice were immunized with DNP-KLH in adjuvant on day 0, and boosted on day 21. The graph shows mean (±SEM) DNP-specific IgG levels as determined by ELISA. Images on the right represent immunofluorescence staining of frozen spleen sections from all genotypes harvested 7 days after the boost phase of DNP-KLH immunization. Merged images show the presence of B220 + B cells (red) and GC GL7 + B220 + B cells (yellow). Enlarged regions from these sections indicate typical GC structures present within the follicles of WT and CD22 −/− mice, and detectable GL7 + B220 + B cells within the follicles of CD154 TG mice, but not in CD154 TG CD22 −/− mice (representative regions are shown for comparison). (A–C) Means significantly different from WT are indicated by asterisks (*p≤0.05, **p≤0.01), and between other indicated groups by crosses (†p

    Journal: PLoS ONE

    Article Title: Amplified B Lymphocyte CD40 Signaling Drives Regulatory B10 Cell Expansion in Mice

    doi: 10.1371/journal.pone.0022464

    Figure Lengend Snippet: Impaired IgG and GC responses in CD154 TG CD22 −/− mice. ( A ) Serum IgM and IgG levels of 4 and 12 mo-old WT, CD22 −/− , CD154 TG , and CD154 TG CD22 −/− mice. Symbols represent serum concentrations for individual mice as determined by ELISA, with means indicated by horizontal bars. ( B ) Serum autoAbs reactive with dsDNA, ssDNA, or histone proteins in 12 mo-old mice. ELISA OD values for IgM (upper panels) and IgG (lower panels) autoAbs are shown for individual mice, with means indicated by horizontal bars. Sera from 2 mo-old WT C57BL/6 and 6 mo-old MLR lpr mice were used as negative and positive controls, respectively. ( C ) Impaired IgG responses to a TD Ag. WT ( n = 3), CD22 −/− ( n = 4), CD154 TG ( n = 6), and CD154 TG CD22 −/− ( n = 8) mice were immunized with DNP-KLH in adjuvant on day 0, and boosted on day 21. The graph shows mean (±SEM) DNP-specific IgG levels as determined by ELISA. Images on the right represent immunofluorescence staining of frozen spleen sections from all genotypes harvested 7 days after the boost phase of DNP-KLH immunization. Merged images show the presence of B220 + B cells (red) and GC GL7 + B220 + B cells (yellow). Enlarged regions from these sections indicate typical GC structures present within the follicles of WT and CD22 −/− mice, and detectable GL7 + B220 + B cells within the follicles of CD154 TG mice, but not in CD154 TG CD22 −/− mice (representative regions are shown for comparison). (A–C) Means significantly different from WT are indicated by asterisks (*p≤0.05, **p≤0.01), and between other indicated groups by crosses (†p

    Article Snippet: C57BL/6 WT control mice were purchased from either The Jackson Laboratory (Bar Harbor, ME) or NCI Frederick (Bethesda, MD).

    Techniques: Mouse Assay, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining

    LTB 4 is required for IL-1β production. A) Quantification of IL-1β and B) Il1b mRNA expression in the skin of C57BL/6, Ltb4r1 −/− , C57BL/6 treated with a topical ointment containing LTB 4 or C57BL/6 treated with a topical ointment containing BLT1 antagonist U-75302 24h after MRSA infection. C) IVIS scanning of pIL1DsRed mice treated with a topical ointment containing BLT1 antagonist U-75032 or vehicle. TNF-α quantification in D) peritoneal macrophages treated with LTB 4 or BLT1 antagonist before MRSA infection and E) skin biopsy from wild-type (WT) or Ltb4r1 −/− mice after 24h MRSA infection. F) IL-1β quantification from human neutrophils isolated from healthy blood donors treated with LTB 4 and LPS. Data are the mean ± SEM of 3-10 mice. *p

    Journal: bioRxiv

    Article Title: Leukotriene B4 licenses inflammasome activation to enhance skin host defense

    doi: 10.1101/2020.02.03.932129

    Figure Lengend Snippet: LTB 4 is required for IL-1β production. A) Quantification of IL-1β and B) Il1b mRNA expression in the skin of C57BL/6, Ltb4r1 −/− , C57BL/6 treated with a topical ointment containing LTB 4 or C57BL/6 treated with a topical ointment containing BLT1 antagonist U-75302 24h after MRSA infection. C) IVIS scanning of pIL1DsRed mice treated with a topical ointment containing BLT1 antagonist U-75032 or vehicle. TNF-α quantification in D) peritoneal macrophages treated with LTB 4 or BLT1 antagonist before MRSA infection and E) skin biopsy from wild-type (WT) or Ltb4r1 −/− mice after 24h MRSA infection. F) IL-1β quantification from human neutrophils isolated from healthy blood donors treated with LTB 4 and LPS. Data are the mean ± SEM of 3-10 mice. *p

    Article Snippet: Eighteen-week-old female or male BLT1−/− (B6.129S4-Ltb4r1tm1Adl /J , LysMcre, MMDTR, and strain-matched WT C57BL/6 mice were purchased from Jackson Labs (Bar Harbor, ME USA).

    Techniques: Expressing, Infection, Mouse Assay, Isolation

    Protective efficacy of anti-JEV MAbs in mice. (A and B) Four- to 5-week-old male C57BL/6 mice were passively administered 10 µg of the indicated (A) mouse or (B) human MAb via intraperitoneal injection 1 day prior to inoculation with 10 2 FFU of JEV-Nakayama via the subcutaneous route. JEV-31 ( n = 9), JEV-106 ( n = 8), JEV-143 ( n = 8), and JEV-169 ( n = 10) provided complete protection against lethality. JEV-27 ( n = 8), JEV-128 ( n = 9), and JEV-131 ( n = 9) provided partial protection compared to the isotype control MAbs. (C and D) Three-week-old male C57BL/6 mice were passively administered 10 µg of the indicated MAb as described above 1 day prior to inoculation with 10 3 FFU of (C) JEV-MAR 859 (JEV-31, n = 8; JEV-131, n = 9; JEV-169, n = 8) or (D) JEV-2372/79 (JEV-31, n = 9; JEV-106, n = 9; JEV-131, n = 9; JEV-169, n = 9). (E and F) Two hundred fifty micrograms of the indicated MAb was administered 5 days postinfection to (E) 4- to 5-week-old mice infected with 10 2 FFU of JEV-Nakayama (JEV-31, n = 9; JEV-106, n = 9; JEV-143, n = 9; JEV-169, n = 9; hJEV-75, n = 8) or (F) 3-week-old mice infected with 10 3 FFU of JEV-2372/79 (JEV-31, n = 10; JEV-131, n = 9; JEV-143, n = 9; JEV-169, n = 10; hJEV-75, n = 9). Data are pooled from at least two independent experiments. Survival was analyzed for each MAb compared to the isotype control MAb by the log rank test. *, P

    Journal: mBio

    Article Title: Mouse and Human Monoclonal Antibodies Protect against Infection by Multiple Genotypes of Japanese Encephalitis Virus

    doi: 10.1128/mBio.00008-18

    Figure Lengend Snippet: Protective efficacy of anti-JEV MAbs in mice. (A and B) Four- to 5-week-old male C57BL/6 mice were passively administered 10 µg of the indicated (A) mouse or (B) human MAb via intraperitoneal injection 1 day prior to inoculation with 10 2 FFU of JEV-Nakayama via the subcutaneous route. JEV-31 ( n = 9), JEV-106 ( n = 8), JEV-143 ( n = 8), and JEV-169 ( n = 10) provided complete protection against lethality. JEV-27 ( n = 8), JEV-128 ( n = 9), and JEV-131 ( n = 9) provided partial protection compared to the isotype control MAbs. (C and D) Three-week-old male C57BL/6 mice were passively administered 10 µg of the indicated MAb as described above 1 day prior to inoculation with 10 3 FFU of (C) JEV-MAR 859 (JEV-31, n = 8; JEV-131, n = 9; JEV-169, n = 8) or (D) JEV-2372/79 (JEV-31, n = 9; JEV-106, n = 9; JEV-131, n = 9; JEV-169, n = 9). (E and F) Two hundred fifty micrograms of the indicated MAb was administered 5 days postinfection to (E) 4- to 5-week-old mice infected with 10 2 FFU of JEV-Nakayama (JEV-31, n = 9; JEV-106, n = 9; JEV-143, n = 9; JEV-169, n = 9; hJEV-75, n = 8) or (F) 3-week-old mice infected with 10 3 FFU of JEV-2372/79 (JEV-31, n = 10; JEV-131, n = 9; JEV-143, n = 9; JEV-169, n = 10; hJEV-75, n = 9). Data are pooled from at least two independent experiments. Survival was analyzed for each MAb compared to the isotype control MAb by the log rank test. *, P

    Article Snippet: WT C57BL/6 male mice (4 to 5 weeks old; Jackson Laboratories) were inoculated with 102 FFU of JEV-Nakayama subcutaneously in the footpad.

    Techniques: Mouse Assay, Injection, Infection