Journal: PLoS ONE

Article Title: Networked T Cell Death following Macrophage Infection by Mycobacterium tuberculosis

doi: 10.1371/journal.pone.0038488

Figure Lengend Snippet: (A) Addition of the pan-caspase inhibitor Z-VAD-FMK mitigated cell death induced by low MOI and high MOI-infected macrophage supernatants (shaded bars, infection + and ++ respectively), as well as background cell death observed in the uninfected control supernatants (closed bars, infection -). Cell death in infected supernatants was not mitigated by coincubation with blocking antibodies to TNF-α (B) or Fas (C), and in each case was significantly higher than in uninfected control supernatants (closed bars). Cell death induced by positive controls (D) (1 µM Staurosporine, 5 ng/ml recombinant human TNF-α +0.2 µg/ml cycloheximide and 10 ng/ml recombinant human Fas ligand) was in each case significantly reduced by coincubation with the corresponding inhibitor (open bars). Data shown in each panel are from one representative experiment, showing mean percentage cell death ± SEM from triplicate incubations. ** = p<0.01, *** = p<0.001, Student’s t test relative to uninfected control (A–C), or positive control relative to inhibitor (D).

Article Snippet: In positive control experiments for Fas ligand-mediated cell killing, cells were incubated in the presence of 10 ng/ml recombinant human Fas ligand (R&D systems), crosslinked with 10 μg/ml anti-6× Histidine (R&D systems).

Techniques: Infection, Control, Blocking Assay, Recombinant, Positive Control

Journal:

Article Title: Threshold levels of Flt3-ligand are required for the generation and survival of lymphoid progenitors and B cell precursors

doi: 10.1002/eji.201040710

Figure Lengend Snippet: Monoallelic expression of FL reduces FL production. (A) Semi-quantitative RT-PCR of FL transcripts in BM cells from C57BL/6 (B6), FL+/+ x RAG1-GFP/+, FL+/− x RAG1-GFP/+, or FL−/− x RAG1-GFP/+ mice. The cDNA was serially diluted 1:1, 1:3, and 1:9 for semi-quantitative analysis. Beta-actin was used as a loading control. Data are representative of two BM samples for each genotype. (B) Quantification of FL transcripts. Intensity data are the average of two BM samples for each genotype. (C) Concentration of FL (pg/mL) in the serum of C57Bl/6 (B6), FL+/−, and FL−/− mice as determined by ELISA. Data represents the mean ± S.D. (*p ≤ 0.0001) FL concentration in serum from ≥ 5 mice/genotype and 2 independent experiments. P-values were determined using the Students T-test.

Article Snippet: The serum concentration of FL was calculated by ELISA on serum harvested from FL+/+ , FL+/− , and FL−/− mice using mouse Flt3 Ligand Quantikine ELISA Kit (R&D Systems, Minneapolis, MN) per kit instructions.

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

Journal:

Article Title: Threshold levels of Flt3-ligand are required for the generation and survival of lymphoid progenitors and B cell precursors

doi: 10.1002/eji.201040710

Figure Lengend Snippet: FL haploinsufficiency reduces LHP and RAG1 locus activation. (A) Flow cytometric analysis of LSK+ BM cells from RAG1-GFP x FL+/+, RAG1-GFP x FL+/−, and RAG1-GFP x FL−/− mice (top panels). Differential expression of Flt3 and VCAM-1 in LSK+ BM cells to discriminate HSC/MPP, GMLP, and LMPP (bottom panels). Boxed regions indicate Flt3hi GMLP. (B) Histogram of boxed region from (A) bottom panels, indicative of Flt3 expression in Flt3hi GMLP in different FL genotypes. (C) Flow cytometric analysis of LSK+ BM cells from RAG1-GFP+ x FL mice to distinguish HSC, MPP, and LHP. A littermate GFP- control was analyzed in each experiment to determine GFP+ gates (data not shown). (D) Flow cytometric analysis of Lin- BM cells to distinguish CLP: Lin- c-kitlo IL-7R+ (Top panels). CLP were further discriminated by Flt3 and Sca-1 expression (middle panels). GFP expression within Lin- c-kitlo IL-7R+ Flt3+ Sca-1+ CLP (bottom panels). GFP+ gates were determined by analysis of Lin- c-kitlo IL-7R- Flt3+ cells which do not express GFP (data not shown). (E) Flow cytometric analysis of BCP. Pre-Pro-B/Pro-B cells are B220+ CD43+, Pre-B cells are B220+ CD43-, and naïve/mature B cells are B220hi CD43-. Data are representative of ≥ 5 mice/genotype and ≥ 3 independent experiments.

Article Snippet: The serum concentration of FL was calculated by ELISA on serum harvested from FL+/+ , FL+/− , and FL−/− mice using mouse Flt3 Ligand Quantikine ELISA Kit (R&D Systems, Minneapolis, MN) per kit instructions.

Techniques: Activation Assay, Quantitative Proteomics, Expressing, Control

Journal:

Article Title: Threshold levels of Flt3-ligand are required for the generation and survival of lymphoid progenitors and B cell precursors

doi: 10.1002/eji.201040710

Figure Lengend Snippet: Id1 does not rescue the lymphoid defect in FL−/− mice. (A) Flt3, tcfe2a, rag1, and ebf1 and (B) Scl and id1 were analyzed from FACS sorted GMLP and LMPP from C57Bl/6 (B6) and FL+/− mice by quantitative PCR. (A+B) Data are representative of 2 independent experiments with BM pooled from ≥ 5 mice/genotype. Error bars represent the mean ± SEM. (C) Flow cytometric analysis of BM from C57Bl/6 (B6), Id1−/−, FL−/−, and FL−/− Id1−/− mice stained with antibodies for lineage markers, c-kit, and Sca-1 to determine LSK+ cells (top panels). LSK+ cells stained with antibodies to Flt3 and VCAM-1 to distinguish HSC/MPP, GMLP, and LMPP (bottom panels). Data are representative of ≥ 3 mice/genotype and ≥ 3 independent experiments.

Article Snippet: The serum concentration of FL was calculated by ELISA on serum harvested from FL+/+ , FL+/− , and FL−/− mice using mouse Flt3 Ligand Quantikine ELISA Kit (R&D Systems, Minneapolis, MN) per kit instructions.

Techniques: Real-time Polymerase Chain Reaction, Staining

Journal:

Article Title: Threshold levels of Flt3-ligand are required for the generation and survival of lymphoid progenitors and B cell precursors

doi: 10.1002/eji.201040710

Figure Lengend Snippet: Flt3 regulates the survival, but not the proliferation of LHP. (A-B) Flow cytometric analysis of bone marrow to examine BrdU incorporation 12 hours after 2 mg were injected i.p. into FL+/+ and FL−/− mice in c-kit+ Sca-1+ (SK+) (A) and c-kit+ Sca-1- (B). (A) Total BrdU incorporation in SK+ cells (top panels). BrdU incorporation in SK+ cells using Flt3 as additional criteria (bottom panels). (B) Total BrdU incorporation in c-kit+ Sca-1- cells (top panels). BrdU incorporation in c-kit+ Sca-1- cells using Flt3 as additional criteria (bottom panels). BrdU quadrants are set on the IgG isotype control for each genotype (data not shown). Data are representative of ≥ 3 mice/genotype and ≥ 3 independent experiments. (C-D) Annexin V staining and intracellular Mcl-1 expression in LSK+ cells (C) and CLP (D). FL+/+ represented by filled histogram, FL−/− indicated by solid line, and thin line depicts isotype control. (C) LSK+ cells were fractionated into Flt3lo and Flt3+hi subsets (top panels). Overlaid histograms depicting Annexin V staining in LSK+ Flt3lo and LSK+ Flt3+hi cells (middle panels). Overlaid histograms depicting intracellular Mcl-1 staining in LSK+ Flt3lo and LSK+ Flt3+hi cells (bottom panels). (D) CLP fractionated into three subsets: Flt3neg, Flt3lo, and Flt3hi (top panels). Overlaid histograms depicting Annexin V staining in CLP subsets (middle panels). Overlaid histograms depicting intracellular Mcl-1 staining in CLP subsets (bottom panels). Data are representative of 3 independent experiments (C+D).

Article Snippet: The serum concentration of FL was calculated by ELISA on serum harvested from FL+/+ , FL+/− , and FL−/− mice using mouse Flt3 Ligand Quantikine ELISA Kit (R&D Systems, Minneapolis, MN) per kit instructions.

Techniques: BrdU Incorporation Assay, Injection, Control, Staining, Expressing