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

Journal: Immunity

Article Title: Activation of p53 in immature myeloid precursor cells controls differentiation into Ly6c + CD103 + monocytic antigen-presenting cells in tumors

doi: 10.1016/j.immuni.2017.12.014

Figure Lengend Snippet: Key Resources Table

Article Snippet: Tumors were disaggregated by treating for 1 hr with collagenase, DNAse and hyaluronidase in RPMI 1640 medium, as described ( Sharma et al., 2015 ). table ft1 table-wrap mode="anchored" t5 REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies anti-Flt3L neutralizing antibody (goat polyclonal) R&D Systems Cat#AF427 anti-IL-12p40 neutralizing antibody (clone C17.8) BioXcell Cat#BE0051 anti-CTLA4 blocking antibody (clone 9D9) BioXcell anti-PD-1 blocking antibody (clone J43) Laboratory of Dr. Hideo Yagita, or from BioXcell ( Agata et al., 1996 ), Cat#BP0033-2 anti-PDL2 blocking antibody (clone TY25) Laboratory of Dr. Hideo Yagita, or from BioXcell ( Yamazaki et al., 2002 ), Cat#BE0112 anti-PD-L1 blocking antibody (clone MIH7) Laboratory of Dr. Miyuki Azuma ( Tsushima et al., 2003 ) CD4 (mouse) (clone RM4-5) BD-Bioscience Cat#553051 CD8 (mouse) (clone 53-6.7) BD-Bioscience Cat# 561092 CD86 (mouse) (clone GL1) BD-Bioscience Cat#553692 CD11c (clone HL3) BD-Bioscience Cat#561119 Ly6c (mouse) (clone AL-21) BD-Bioscience Cat#560592 IFNγ (mouse) (clone XMG1.2) BD-Bioscience Cat#554412 CD24 (clone M1/69) BD-Bioscience Cat#561079 CCR7 (mouse) (clone 4B12) BD-Bioscience Cat#560682 Foxp3 (mouse) (clone FJK-16s) eBioscience Cat#11-5773 granzyme B (mouse) (clone NGZB) eBioscience Cat#12-8898 PD1 (mouse) (Clone: J43) eBioscience Cat#12-9985 PDL1 (mouse) (clone MIH5) eBioscience Cat#12-5982 CD103 (mouse) (M290) BD-Bioscience Cat#557495 Ly6c (mouse) (clone HK1.4) eBioscience Cat#45-5932 CD69 (mouse) (clone H1.2F3) eBioscience Cat#11-0691 NOS2 (mouse) (C-11) Santa Cruz Biotech.

Techniques: Blocking Assay, Virus, Recombinant, Adjuvant, Isolation, Software