News Article | May 9, 2017
As part of the Protein and Cell Analysis Education Series, which provides free-access to a series of educational webinars, Thermo Fisher Scientific has announced the addition of an Antibody Validation Forum. Attendees will learn from a panel discussion with key scientific leaders as they address the antibody reproducibility crisis. Reproducibility and antibody validation standards are two significant challenges facing scientific researchers today. To address these issues, Thermo Fisher Scientific, a leading producer of antibodies, supported a group of leading researchers from several global institutions. This International Working Group for Antibody Validation (IWGAV) brainstormed and developed a set of proposed standards to optimize antibody validation methods with a specific emphasis on antibody specificity verification. In this event, Thermo Fisher Scientific will host a live, virtual round table discussion in which representative IWGAV members and others from the research community will participate in a dialogue about antibody validation standards and practices, with the goal of finding consensus for how to further methods for verifying antibodies target specificity, using tests such as Knockout, Knockdown, IP-Mass Spectometry and Cell Treatment. LabRoots will host the forum May 30, 2017, beginning at 9:00 a.m. PDT, with additional presentations to follow. For more information on the entire Protein and Cell Analysis Education Series, this event along with complete agenda of webinars and biographies on the speakers, or to register for free, click here. *The use or any variation of the word “validation” refers only to research use antibodies that were subject to functional testing to confirm that the antibody can be used with the research techniques indicated. It does not ensure that the product(s) was validated for clinical or diagnostic uses. For Research Use Only. Not for use in diagnostic procedures. ©2017. Thermo Fisher Scientific, Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. We hereby disclose that this email communication is for commercial purposes. About Thermo Fisher Scientific Thermo Fisher Scientific Inc. is the world leader in serving science, with revenues of $17 billion and more than 50,000 employees in 50 countries. Our mission is to enable our customers to make the world healthier, cleaner and safer. We help our customers accelerate life sciences research, solve complex analytical challenges, improve patient diagnostics and increase laboratory productivity. Through our premier brands – Thermo Scientific, Applied Biosystems, Invitrogen, Fisher Scientific and Unity Lab Services – we offer an unmatched combination of innovative technologies, purchasing convenience and comprehensive support. For more information, please visit http://www.thermofisher.com. ABOUT LABROOTS LabRoots is the leading scientific social networking website and producer of educational virtual events and webinars. Contributing to the advancement of science through content sharing capabilities, LabRoots is a powerful advocate in amplifying global networks and communities. Founded in 2008, LabRoots emphasizes digital innovation in scientific collaboration and learning, and is a primary source for current scientific news, webinars, virtual conferences, and more. LabRoots has grown into the world’s largest series of virtual events within the Life Sciences and Clinical Diagnostics community.
News Article | May 10, 2017
To generate CRISPR–Cas9 plasmids targeting the last exon of LGR5 (exon 18) or KRT20 (exon 8), 20-bp target sequences were cloned into a pX330-U6-Chimeric_BB-CBh-hSpCas9 plasmid (Addgene 42230) to obtain single vectors bicistronically expressing sgRNA and human codon-optimized Cas9 nuclease as previously described36. The sgRNA sequences targeting LGR5 or KRT20 are available in Supplementary Table 1. To construct donor vectors for LGR5–GFP- and KRT20–GFP-knock-in, 5′ and 3′ homology arms (1 kbp each) were amplified by PCR and cloned into an Ires-GFP-loxp-pEF1α-RFP-T2A-puro-loxp plasmid (HR180PA-1, SBI) using the In-Fusion HD Cloning kit (Clontech). For CreER or iCaspase9-T2A-tdTomato knock-in, we replaced GFP of the LGR5–GFP or KRT20–GFP construct with CreER or iCaspase9-T2A-tdTomato, respectivley. The final plasmid sequences were verified by DNA sequencing. To obtain a rainbow reporter, the rainbow cassette was excised from a CMV-Brainbow-2.1R plasmid (Addgene 18723) and cloned into a PiggyBac vector (PB510B-1, SBI). For bioluminescent imaging, we cloned optimized firefly luciferase luc2 into a GFP-expressing PiggyBac vector (PB513B-1, SBI). Knock-in efficiency and diver mutation profiles for each CCO line are available in Supplementary Table 2. All organoids were established as previously reported16 from patients who had given informed consent under the ethical committee of Keio University School of Medicine. The organoids were embedded in Matrigel and cultured with previously described basal culture medium37, specifically Advanced Dulbecco’s modified Eagle’s medium/F12 supplemented with penicillin/streptomycin, 10 mM HEPES, 2 mM GlutaMAX, 1× B27 (Life Technologies), 10 nM gastrin I (Sigma) and 1 mM N-acetylcysteine (Sigma). The following niche factors were added to the basal culture medium depending on the niche requirements of CRC organoid lines: 50 ng ml−1 mouse recombinant EGF, 100 ng ml−1 mouse recombinant noggin (PeproTech) and 500 nM A83-01 (Tocris). We electroporated the vectors under previously reported conditions37. Three days after electroporation, the organoids were selected with puromycin (2 μg ml−1) treatment for two days. For in vitro ablation experiments, we treated the organoids with 1 nM dimerizer (AP20187, Clontech). Drug-resistant organoid clones were manually selected and expanded individually. Genomic DNA was isolated using the QIAamp DNA blood mini kit (Qiagen). Legitimate knock-in was determined by PCR. Southern blotting was performed based on the standard procedure using 1 μg of genomic DNA. The sequences of PCR primers and Southern blot probes are shown in Supplementary Table 1. The puromycin-RFP selection cassette flanked by loxP sequences was excised by transient infection of Cre-expressing adenovirus (TaKaRa) at multiplicity of infection of 5–10. After the infection, we manually selected and cloned RFP− organoids. Deletion of the puromycin cassette was validated by PCR diagnostics. Percentage of successful knock-in for each line is shown in Supplementary Table 2. Once a knock-in reporter CCO was cloned, we used the same clone for further experiments. Organoids were dissociated into single cells with TrypLE Express (Life Technology), and large clusters were removed with a CellTrics 20-μm cell strainer (Partec). The cells were washed with cold PBS and stained with 7-amino-actinomycin D (7-AAD) staining solution (BD Biosciences) to exclude dead cells. Single cells were gated based on the SSC-H versus SSC-W profile. The cells were subsequently analysed using a flow cytometer with a 70-μm nozzle (FACS JAZZ, BD Biosciences). Then, 1,000 sorted cells were embedded in 25 μl of Matrigel and cultured in a 48-well plate for 7–10 days. We added 10 μM Y27632 for the first two days of culture, and the organoid colony formation was assessed using a BZX-700 fluorescence microscope (Keyence). RNA was extracted from 1 × 106 sorted cells using the RNeasy Plus mini kit (Qiagen). The RNA quality was determined by the RNA integrity number (RIN) value with the RNA6000 assay (Agilent). Only specimens with RIN > 7.0 were used in this study. Gene expression was determined by microarray (GeneChip PrimeView Human Gene Expression Array, Affymetrix) according to the manufacturer’s instructions. The data were normalized using the robust multi-array analysis implemented in the R package affy. The probes were summarized into genes by selecting probes with the highest median absolute deviation value per gene. GSEA was performed using gsea (in the R package phenoTest) with 1,000 permutations. Two independent intestinal stem cell signature gene sets from refs 17, 38 were used. All animal procedures were approved by the Keio University School of Medicine Animal Care Committee. NOD/Shi-scid,IL-2Rγnull (NOG) mice39 (7–12 weeks of age, male) were obtained from the Central Institute for Experimental Animals (CIEA, Japan). Organoids with the indicated genetic reporter and with or without GFP-luc2-reporter, equivalent to 1 × 105 cells, were xenotransplanted subcutaneously or into the renal subcapsules as previously described40. We monitored the tumour size with a calliper or through bioluminescence imaging. Tumour volumes were measured according to the formula (length × width2) / 2. Once any individual tumour reached 2 cm in size, the mouse was euthanized. For bioluminescence imaging, we intraperitoneally administered 3 mg of D-luciferin (SPI, Tokyo) to tumour-bearing mice 10–20 min before imaging and anaesthetized the animals with isoflurane. The bioluminescence signal was measured with an IVIS imaging system (Xenogen), and the specific signal was calculated as the ratio of photon counts from the region of interest to counts from a background region. The grafts were fixed for subsequent histological analyses. An investigator blinded to the experimental conditions measured the tumour sizes. For the lineage-tracing experiments, each mouse received a single intraperitoneal injection of 0.25 mg (clonal dose) or 1 mg of tamoxifen (Sigma-Aldrich) diluted in corn oil. For the ablation studies, 40 μg of dimerizer was administered for five days daily for short term ablation and on alternate days for long term ablation. To label the proliferating cells, we intraperitoneally administered BrdU (40 mg kg−1, BD Biosciences) and EdU (10 mg kg−1, Life Technologies) at the indicated times. For chemotherapeutic studies, CTX (40 mg kg−1, Merck Serono) or oxaliplatin (15 mg kg−1, AdooQ Bioscience) was administered intraperitoneally at the indicated times. We isolated tumours from xenografted mice and immediately fixed them with 4% paraformaldehyde. Eight-micrometre OCT frozen tissue sections or 5-μm paraffin-embedded tissue sections were processed using a standard histological protocol. For rainbow fluorescent imaging, the frozen sections were visualized using an SP8 confocal microscope (Leica) with the following settings: mCFP was excited at 405 nm and collected using a 480–485-nm filter, nuclear GFP was excited at 488 nm and collected using a 494–507-nm filter, EYFP was excited at 514 nm and collected using a 560–566-nm filter, and RFP was excited at 552 nm and collected using a 601–665-nm filter. Nuclei were counterstained with the near-infrared nuclear dye DRAQ5 (BioStatus). For DLS 3D imaging, the whole tumours were cut into 1–2 mm3 pieces, fixed and embedded in agarose gel. 3D images were acquired using the Leica SP8 DLS system. The proportion of surviving clones was determined by counting the number of RFP+ cells at day 3 and day 31 after tamoxifen administration. Clone identification and raw volume measurement were carried out automatically using the ImageJ 3D-image processing package ‘3D object counter’41, 42. False identification rate of this automatic measurement was determined manually by random sampling. Raw volume was adjusted by randomly subtracting a proportion of clones according to the false-rate. The threshold volume for total colonies on day 3 was set as <2 × 105 μm3 and for large colonies on day 31 as >2 × 105 μm3 (equivalent to 20 cells). Colony-formation efficiency was defined as the ratio of the number of large colonies on day 31 to the number of clones on day 3. For immunostaining, the following primary antibodies were used: mouse anti-cytokeratin-20 (M7019, clone K 20.8, Dako, 1:50), goat anti-GFP (ab6673, Abcam, 1:200), rabbit anti-Ki67 (ab16667, Abcam, 1:100), mouse anti-α smooth muscle actin ab-1 (MS-113-P, Thermo Scientific, 1:800), mouse anti-BrdU (347580, BD, 1:100), anti-cleaved caspase-3 (9661, Cell Signaling, 1:100) and anti-tdTomato (600-401-379, ROCKLAND, 1:500). Alexa Fluor 488-, 568- or 647-conjugated secondary antibodies (Life Technologies, donkey anti-mouse, rabbit, rat or goat antibodies) were used at 1:200 dilution. For EdU staining, we used the Click-IT Plus EdU Imaging kit (Life Technologies) according to the manufacturer’s instructions. Nuclei were counterstained with Hoechst 33342 or DAPI. Images were captured with a Leica SP8 confocal microscope or a BZX-700 fluorescence microscope (Keyence). To count the number of BrdU/EdU-stained cells, we used Imaris (Bitplane). In situ hybridization was performed using an RNAscope 2.5HD kit (Advanced Cell Diagnostics) according to the manufacturer’s instructions. For each experiment, we used PPIB and DapB genes as positive and negative controls, respectively. Tumour tissues were homogenized using TissueLyser LT (Qiagen) and RNA was extracted with the RNAeasy mini kit (Qiagen) according to the manufacturer’s instructions. cDNA was synthesized using the Omniscript RT kit (Qiagen). Quantitative real-time PCR was performed on LightCycler 96 (Roche Diagnostics) using FastStart Essential DNA Probes Master (Roche Diagnostics) and the cDNAs as templates. Relative LGR5 expression to ACTB was calculated based on the comparative C method. Primers and probes for LGR5 and ACTB are available in Supplementary Table 1. The sample size was determined by previous experience and preliminary experiments. The vehicle/dimerizer/chemotherapy-treated group was randomly assigned on the basis of tumour size at the time of injection. Appropriate statistical analyses were performed dependent on the comparisons referenced in the figure legends. The n values represent biological replicates. All graphs show mean and error bars represent the standard error of the mean (s.e.m.). Genetic mutation data of organoids are summarized in Supplementary Table 2 and described in ref. 16. The microarray dataset generated in this study is available in the Gene Expression Omnibus (accession number: GSE83513). All other data are available from the corresponding author upon reasonable request.
News Article | May 10, 2017
Patients who were included in the study all had Goodpasture disease and fulfilled the following key diagnostic criteria: (1) serum anti-α3(IV)NC1 IgG by enzyme-linked immunosorbent assay (ELISA), (2) linear IgG staining of the GBM and (3) necrotizing and crescentic glomerulonephritis. HLA-DR15 typing of patients was done by monoclonal antibody staining (BIH0596, One Lambda) and flow cytometry. Blood from HLA-typed healthy humans was collected via the Australian Bone Marrow Donor Registry. HLA-DR15, HLA-DR1 and HLA-DR15/DR1 donors were molecularly typed and were excluded if they expressed DQB1*03:02, which is potentially weakly associated with susceptibility to anti-GBM disease2. Studies were approved by the Australian Bone Marrow Donor Registry and Monash Health Research Ethics Committees, and informed consent was obtained from each individual. Mouse MHCII deficient, DR15 transgenic mice and mouse MHCII deficient, DR1 transgenic mice were derived from existing HLA transgenic colonies and intercrossed so that they were on the same background as previously described4. The background was as follows: 50% C57BL/10, 43.8% C57BL/6, 6.2% DBA/2; or with an Fcgr2b−/− background: 72% C57BL/6, 25% C57BL/10 and 3% DBA/2. To generate mice transgenic for both HLA-DR15 and HLA-DR1, mice transgenic for either HLA-DR15 or HLA-DR1 were intercrossed. FcγRIIb intact HLA transgenic mice and cells were used for all experiments, except those in experimental Goodpasture disease, where Fcgr2b−/− HLA transgenic strains were used. While DR15+ mice readily break tolerance to α3(IV)NC1 when immunized with human α3 or mouse α3 , renal disease is mild4. As genetic changes in fragment crystallizable (Fc) receptors have been implicated in the development of nephritis in rodents and in humans18, Fcgr2b−/− HLA transgenic strains were used when end organ injury was an important endpoint. For in vitro experiments, cells from either male or female mice were used. For in vivo experiments both male and female mice were used, for immunization aged 8–12 weeks and for the induction of experimental Goodpasture disease aged 8–10 weeks. Experiments were approved by the Monash University Animal Ethics Committee (MMCB2011/05 and MMCB2013/21). HLA-DR15-α3 and HLA-DR1-α3 were produced in High Five insect cells (Trichoplusia ni BTI-Tn-5B1-4 cells, Invitrogen) using the baculovirus expression system essentially as described previously for HLA-DQ2/DQ8 proteins19, 20. Briefly, synthetic DNA (Integrated DNA Technologies, Iowa, USA) encoding the α- and β-chain extracellular domains of HLA-DR15 (HLA-DR1A*0101, HLA-DRB1*15:01), HLA-DR1 (HLA-DR1A*0101, HLA-DRB1*01:01) and the α3 peptide were cloned into the pZIP3 baculovirus vector19, 20. To promote correct pairing, the carboxy (C) termini of the HLA-DR15 and HLA-DR1 α- and β-chain encoded enterokinase cleavable Fos and Jun leucine zippers, respectively. The β-chains also encoded a C-terminal BirA ligase recognition sequence for biotinylation and a poly-histidine tag for purification. HLA-DR15-α3 and HLA-DR1-α3 were purified from baculovirus-infected High Five insect cell supernatants through successive steps of immobilized metal ion affinity (Ni Sepharose 6 Fast-Flow, GE Healthcare), size exclusion (S200 Superdex 16/600, GE Healthcare) and anion exchange (HiTrap Q HP, GE Healthcare) chromatography. For crystallization, the leucine zipper and associated tags were removed by enterokinase digestion (Genscript, New Jersey, USA) further purified by anion exchange chromatography, buffer exchanged into 10 mM Tris, pH 8.0, 150 mM NaCl and concentrated to 7 mg ml−1. Purified HLA-DR15-α3 and HLA-DR1-α3 proteins were buffer exchanged into 10 mM Tris pH 8.0, biotinylated using BirA ligase and tetramers assembled by addition of Streptavidin-PE (BD Biosciences) as previously described19. In mice, 107 splenocytes or cells from kidneys were digested with 5 mg ml−1 collagenase D (Roche Diagnostics, Indianapolis, Indiana, USA) and 100 mg ml−1 DNase I (Roche Diagnostics) in HBBS (Sigma-Aldrich) for 30 min at 37 °C, then filtered, erythrocytes lysed and the CD45+ leukocyte population isolated by MACS using mouse CD45 microbeads (Miltenyi Biotec); they were then surface stained with Pacific Blue-labelled anti-mouse CD4 (BD), antigen-presenting cell (APC)-Cy7-labelled anti-mouse CD8 (BioLegend) and 10 nM PE-labelled tetramer. Cells were then incubated with a Live/Dead fixable Near IR Dead Cell Stain (Thermo Scientific), permeabilized using a Foxp3 Fix/Perm Buffer Set (BioLegend) and stained with Alexa Fluor 647-labelled anti-mouse Foxp3 antibody (FJK16 s). To determine Vα2 and Vβ6 usage, cells were stained with PerCP/Cy5.5 anti-mouse Vα2 (B20.1, Biolegend) and antigen-presenting cell labelled anti-mouse Vβ6 (RR4-7, Biolegend). For each mouse a minimum of 100 cells were analysed. The tetramer+ gate was set on the basis of the CD8+ population. In humans, 3 × 107 white blood cells were surface stained with BV510-labelled anti-human CD3 (BioLegend), Pacific Blue-labelled anti-human CD4 (BioLegend), PE-Cy7-labelled anti-human CD127 (BioLegend), FITC-labelled anti-human CD25 (BioLegend) and 10 nM PE-labelled tetramer. Then, cells were incubated with a Live/Dead fixable Near IR Dead Cell Stain (Life Technologies), permeabilized using a Foxp3 Fix/Perm Buffer Set (BioLegend) and stained with Alexa Fluor 647-labelled anti-human Foxp3 antibody (150D). The tetramer+ gate was set on the basis of the CD3+CD4− population. As validation controls, we found that HLA-DR1-α3 tetramer+ cells did not bind to HLA-DR1-CLIP tetramers (data not shown). The human α3 peptide (GWISLWKGFSF), the mouse α3 peptide (DWVSLWKGFSF) and control OVA peptide (ISQAVHAAHAEINEAGR) were synthesized at >95% purity, confirmed by high-performance liquid chromatography (Mimotopes). Recombinant murine α3(IV)NC1 was generated using a baculovirus system21 and recombinant human α3(IV)NC1 expressed in HEK 293 cells22. The murine α3(IV)NC1 peptide library, which consists of 28 20-amino-acid long peptides overlapping by 12 amino acids, was synthesized as a PepSet (Mimotopes). To measure peptide specific recall responses, IFN-γ and IL-17A ELISPOTs and [3H]thymidine proliferation assays were used (Mabtech for human ELISPOTs and BD Biosciences for mouse ELISPOTs). To measure pro-inflammatory responses of HLA-DR15-α3 tetramer+ CD4+ T cells in patients with Goodpasture disease, HLA-DR15-α3 tetramer+ CD4+ T cells were enumerated then isolated from peripheral blood mononuclear cells of patients with Goodpasture disease (frozen at the time of presentation) by magnetic bead separation (Miltenyi Biotec) then co-cultured at a frequency of 400 HLA-DR15-α3 tetramer+ CD4+ T cells per well with 2 × 106 HLA-DR15-α3 tetramer-depleted mitomycin C-treated white blood cells and stimulated with either no antigens, α3 (10 μg ml−1) or whole recombinant human α3(IV)NC1 (10 μg ml−1) in supplemented RPMI media (10% male AB serum, 2 mM l-glutamine, 50 μM 2-ME, 100 U ml−1 penicillin and 0.1 mg ml−1 streptomycin) (Sigma-Aldrich). Cells were cultured for 18 h at 37 °C, 5% CO and the data expressed as numbers of IFN-γ or IL-17A spots per well. To measure pro-inflammatory responses of HLA-DR15-α3 tetramer+ CD4+ T cells in DR15+ transgenic mice, HLA-DR15-α3 tetramer+ CD4+ T cells were enumerated then isolated from pooled spleen and lymph node cells of DR15+ transgenic mice, immunized with mouse α3 10 days previously by magnetic bead separation. They were then co-cultured at a frequency of 400 HLA-DR15-α3 tetramer+ CD4+ T cells per well with 106 HLA-DR15-α3 tetramer-depleted mitomycin C-treated white blood cells and stimulated with either no antigens, mouse α3 (10 μg ml−1), human α3 (10 μg ml−1), whole recombinant mα3(IV)NC1 (10 μg ml−1) or whole recombinant hα3(IV)NC1 (10 μg ml−1) in supplemented RPMI media (10% FCS, 2 mM l-glutamine, 50 μM 2-ME, 100 U ml−1 penicillin and 0.1 mg ml−1 streptomycin). Cells were cultured for 18 h at 37 °C, 5% CO and the data expressed as numbers of IFN-γ or IL-17A spots per well. To determine the immunogenic portions of α3(IV)NC1, mice were immunized subcutaneously with peptide pools (containing α3 amino acids 1–92, 81–164, or 153–233; 10 μg per peptide per mouse), the individual peptide or in some experiments mα3 at 10 μg per mouse in Freund’s complete adjuvant (Sigma-Aldrich). Draining lymph node cells were harvested 10 days after immunization and stimulated in vitro (5 × 105 cells per well) with no antigen, peptide (10 μg ml−1) or whole α3(IV)NC1 (10 μg ml−1) in supplemented RPMI media (10% FCS, 2 mM l-glutamine, 50 μM 2-ME, 100 U ml−1 penicillin and 0.1 mg ml streptomycin). For [3H]thymidine proliferation assays, cells were cultured in triplicate for 72 h with [3H]thymidine added to culture for the last 16 h. To measure human α3 - or mouse α3 -specific responses in CD4+ T cells from naive transgenic mice or blood of healthy humans, we used a modification of a previously published protocol23. One million CD4+ T cells were cultured with 106 mitomycin-treated CD4-depleted splenocytes for 8 days in 96-well plates with or without 100 μg ml−1 of human α3 or mouse α3 . T cells were depleted from mouse cultures by sorting out CD4+CD25+ and in humans by sorting out CD4+CD25hiCD127lo cells using antibodies and a cell sorter. Cytokine secretion was detected in the cultured supernatants by cytometric bead array (BD Biosciences) or ELISA (R&D Systems). To determine proliferation, magnetically separated CD4+ T cells were labelled with CellTrace Violet (CTV; Thermo Scientific) before culture. To measure the expansion of T cells, mice were immunized with 100 μg of α3 emulsified in Freund’s complete adjuvant, then boosted 7 days later in Freund’s incomplete adjuvant. Draining lymph node cells were stained with the HLA-DR15-α3 tetramer, CD3, CD4, CXCR5, PD-1, CD8 and Live/Dead Viability dye. To determine the potency of HLA-DR1-α3 tetramer+ T cells, 106 cells per well of CD4+CD25− T effectors isolated by CD4+ magnetic beads and CD25− cell sorting from naive DR15+DR1+ mice were co-cultured with CD4+CD25+ T cells with or without depletion of HLA-DR1-α3 tetramer+ T cells from DR1+ mice at different concentrations: 0, 12.5 × 103, 25 × 103, 50 × 103 and 100 × 103 cells per well in the presence of 106 CD4-depleted mitomycin C-treated spleen and lymph node cells from DR15+DR1+mice in supplemented RPMI media (10% FCS, 2 mM l-glutamine, 50 μM 2-ME, 100 U ml−1 penicillin and 0.1 mg ml−1 streptomycin) containing 100 μg ml−1 of mouse α3 . To determine proliferation, the CD4+CD25− T effector cells were labelled with CTV before culture. Cells were cultured in triplicate for 8 days in 96-well plates. HLA transgenic mice, on an Fcgr2b−/− background, were immunized with 100 μg of α3 or mα3 subcutaneously on days 0, 7 and 14, first in Freund’s complete, and then in Freund’s incomplete, adjuvant. Mice were killed on day 42. Albuminuria was assessed in urine collected during the last 24 h by ELISA (Bethyl Laboratories) and expressed as milligrams per micromole of urine creatinine. Blood urea nitrogen and urine creatinine were measured using an autoanalyser at Monash Health. Glomerular necrosis and crescent formation were assessed on periodic acid-Schiff (PAS)-stained sections; fibrin deposition using anti-murine fibrinogen antibody (R-4025) and DAB (Sigma); CD4+ T cells, macrophages and neutrophils were detected using anti-CD4 (GK1.5), anti-CD68 (FA/11) and anti-Gr-1 (RB6-8C5) antibodies. The investigators were not blinded to allocation during experiments and outcome assessment, except in histological and immunohistochemical assessment of kidney sections. To deplete regulatory T cells, mice were injected intraperitoneally with 1 mg of an anti-CD25 monoclonal antibody (clone PC61) or rat IgG (control) 2 days before induction of disease. In these experiments, mice were randomly assigned to receive control or anti-CD25 antibodies. Individual DR15-α3 -specific CD4+ T cells were sorted into wells of a 96-well plate. Multiplex single-cell reverse transcription and PCR amplification of TCR CDR3α and CDR3β regions were performed using a panel of TRBV- and TRAV-specific oligonucleotides, as described24, 25. Briefly, mRNA was reverse transcribed in 2.5 μl using a Superscript III VILO cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, Massachusetts, USA) (containing 1× Vilo reaction mix, 1× superscript RT, 0.1% Triton X-100), and incubated at 25 °C for 10 min, 42 °C for 120 min and 85 °C for 5 min. The entire volume was then used in a 25 μl first-round PCR reaction with 1.5 U Taq DNA polymerase, 1× PCR buffer, 1.5 mM MgCl , 0.25 mM dNTPs and a mix of 25 mouse TRAV or 40 human TRAV external sense primers and a TRAC external antisense primer, along with 19 mouse TRBV or 28 human TRBV external sense primers and a TRBC external antisense primer (each at 5 pmol μl−1), using standard PCR conditions. For the second-round nested PCR, a 2.5 μl aliquot of the first-round PCR product was used in separate TRBV- and TRAV-specific PCRs, using the same reaction mix described above; however, a set of 25 mouse TRAV or 40 human TRAV internal sense primers and a TRAC internal antisense primer, or a set of 19 mouse TRBV or 28 human TRBV internal sense primers and a TRBV internal antisense primer, were used. Second-round PCR products were visualized on a gel and positive reactions were purified with ExoSAP-IT reagent. Purified products were used as template in sequencing reactions with internal TRAC or TRBC antisense primers, as described. TCR gene segments were assigned using the IMGT (International ImMunoGeneTics) database26. In mouse experiments, three mice were pooled per HLA and the number of sequences obtained were as follows. For TRAV: DR15, n = 81; DR1 n = 84; for TRBV: DR15, n = 100; DR1 n = 87; for TRAJ: DR15, n = 81; DR1 n = 84; and for TCR beta joining (TRBJ): DR15, n = 100; DR1 n = 87. Red-blood-cell-lysed splenocytes from DR1+ and DRB15+DR1+ mice were sorted on the basis of surface expression of CD4 and CD25 and being either DR1-α3 tetramer positive or negative into three groups: (1) CD4+CD25−HLA-DR1-α3 tetramer− T cells; (2) CD4+CD25+HLA-DR1-α3 tetramer− T cells; and (3) CD4+CD25+HLA-DR1-α3 tetramer+ T cells. A minimum of 1,000 cells were sorted. Immediately after sorting, the RNA was isolated and complementary DNA (cDNA) generated using a Cells to Ct Kit (Ambion) followed by a preamplification reaction using Taqman Pre Amp Master Mix (Applied Biosystems), which preamplified the following cDNAs: Il2ra, Foxp3, Ctla4, Tnfrsf18, Il7r, Sell, Pdcd1, Entpd1, Cd44, Tgfb3, Itgae, Ccr6, Lag3, Lgals1, Ikzf2, Tnfrsf25, Nrp1, Il10. The preamplified cDNA was used for RT–PCR reactions in duplicate using Taqman probes for the aforementioned genes. Each gene was expressed relative to 18S, logarithmically transformed and presented as a heat map. The Epstein-Barr-virus-transformed human B lymphoblastoid cell lines IHW09013 (SCHU, DR15-DR51-DQ6) and IHW09004 (JESTHOM, DR1-DQ5) were maintained in RPMI (Invitrogen) supplemented with 10% FCS, 50 IU ml−1 penicillin and 50 μg ml−1 streptomycin. Confirmatory tissue typing of these cells was performed by the Victorian Transplantation and Immunogenetics Service. The B-cell hybridoma LB3.1 (anti-DR) was grown in RPMI-1640 with 5% FCS at 37 °C and secreted antibody purified using protein A sepharose (BioRad). HLA-DR-presented peptides were isolated from naive DR15+Fcgr2b+/+ or DR1+Fcgr2b+/+ mice. Spleens and lymph nodes (pooled from five mice in each group) or frozen pellets of human B lymphoblastoid cell lines (triplicate samples of 109 cells) were cryogenically milled and solubilized as previously described12, 27, cleared by ultracentrifugation and MHC peptide complexes purified using LB3.1 coupled to protein A (GE Healthcare). Bound HLA complexes were eluted from each column by acidification with 10% acetic acid. The eluted mixture of peptides and HLA heavy chains was fractionated by reversed-phase high-performance liquid chromatography as previously described10. Peptide-containing fractions were analysed by nano-liquid chromatography–tandem mass spectrometry (nano-LC–MS/MS) using a ThermoFisher Q-Exactive Plus mass spectrometer (ThermoFisher Scientific, Bremen, Germany) operated as described previously10. LC–MS/MS data were searched against mouse or human proteomes (Uniprot/Swissprot v2016_11) using ProteinPilot software (SCIEX) and resulting peptide identities subjected to strict bioinformatic criteria including the use of a decoy database to calculate the false discovery rate28. A 5% false discovery rate cut-off was applied, and the filtered data set was further analysed manually to exclude redundant peptides and known contaminants as previously described29. The mass spectrometry data have been deposited in the ProteomeXchange Consortium via the PRIDE30 partner repository with the data set identifier PXD005935. Minimal core sequences found within nested sets of peptides with either N- or C-terminal extensions were extracted and aligned using MEME (http://meme.nbcr.net/meme/), where motif width was set to 9–15 and motif distribution to ‘one per sequence’31. Graphical representation of the motif was generated using IceLogo32. Crystal trials were set up at 20 °C using the hanging drop vapour diffusion method. Crystals of HLA-DR15-α3 were grown in 25% PEG 3350, 0.2 M KNO and 0.1 M Bis-Tris-propane (pH 7.5), and crystals of HLA-DR1-α3 were grown in 23% PEG 3350, 0.1 M KNO , and 0.1 M Bis-Tris-propane (pH 7.0). Crystals were washed with mother liquor supplemented with 20% ethylene glycol and flash frozen in liquid nitrogen before data collection. Data were collected using the MX1 (ref. 33) and MX2 beamlines at the Australian Synchrotron, and processed with iMosflm and Scala from the CCP4 program suite34. The structures were solved by molecular replacement in PHASER35 and refined by iterative rounds of model building using COOT36 and restrained refinement using Phenix37 (see Extended Data Table 2 for data collection and refinement statistics). No statistical methods were used to predetermine sample size. For normally distributed data, an unpaired two-tailed t-test (when comparing two groups). For non-normally distributed data, non-parametric tests (Mann–Whitney U-test for two groups or a Kruskal–Wallis test with Dunn’s multiple comparison) were used. Statistical analyses, except for TCR usage, was by GraphPad Prism (GraphPad Software). For each TCR type/region (TRAV, TRBV, TRAJ, TRBJ), we compared the TCR distribution (frequencies of different TCRs) between DR15 and DR1 using Fisher’s exact test. This was applied both to mice and to human samples. The P values associated with those TCR distributions are indicated above the pie-charts. To correct for multiple testing for individual TCRs, we used Holm’s method. *P < 0.05, **P < 0.01, ***P < 0.001. The data that support the findings of this study are available from the corresponding authors upon request. Self-peptide repertoires have been deposited in the Proteomics Identifications Database archive with the accession code PXD005935. Structural information has been deposited in the Protein Data Bank under accession numbers 5V4M and 5V4N.
News Article | May 11, 2017
Now available in Europe, Asia Pacific, emerging markets and Latin America, the new Thermo Scientific refrigerated incubators utilize powerful compressor technology designed to provide optimal temperature conditions for applications that require thermal stability and uniformity above, around or below the usual ambient laboratory temperature. The systems are also suitable for standard 37 °C incubation applications in warm laboratory environments. Available in both a benchtop and a more spacious floor-standing unit, the new Thermo Scientific RI-150 and RI-250 models of refrigerated incubators have been equipped with key attributes to enable ease-of-use, while facilitating temperature uniformity and precise temperature setting for optimal sample safety and reproducibility of results. Key Applications: The systems are suitable for use in a range of pharmaceutical, life science, water treatment, biological research and microbiology applications, including incubation of bacteria and yeast, water testing, hatching of insects and fish, sample storage at specific temperatures, and biochemical oxygen demand (BOD) testing. Features/Benefits: ● Ease of use – Features include a simple single set-point microprocessor control, a timer for automatic start or shut down, an easy calibration routine and a glass window for easy inspection of samples. ● Temperature uniformity – A silent long-shaft fan and unique airflow system helps ensure even temperature distribution throughout the units for reproducible results. ● Flexibility – Choice of benchtop or floor-standing model. The models include a shelving system with 34 to 66 shelf positions for space flexibility. The large unit has lockable casters for easy set-up and relocation. ● Diversity – Broad temperature range from 4 to 60 °C for a multitude of applications. ● Cost-effectiveness – For a lower initial investment, the RI-150 and RI-250 series of refrigerated incubators provide highly accurate temperate environments. ● Sample safety – An adjustable over- and under-temperature alarm function alerts operators of unexpected temperature deviations and open door. An automatic defrost capability enables uninterrupted long-term operation below ambient temperature. The fan motor is prevented from overheating for additional safety. ● Documentation – A standard access port allows easy insertion of independent monitoring probes. An RS232 interface enables data transfer to a computer (software not included). IQ/OQ documentation and services are also available.
News Article | May 24, 2017
"Il costo era un fattore limitante per i pannelli composti da un gran numero di ampliconi. Per i ricercatori che necessitano di modificare il contenuto genetico di frequente, un prezzo inferiore per gli oligo rappresenta un elemento davvero importante", ha detto Pan Zhang, medico e dottore, direttore presso il Sequencing and Microarray Center (Centro di sequenziamento e micromatrice) del Coriell Institute for Medical Research (Istituto Corriell per la ricerca medica). Adam Ameur, dottore e scienziato bioinformatico per la National Genomics Infrastructure (Infrastruttura genomica nazionale) dello SciLifeLab dell'Università di Uppsala, ha aggiunto: "La maggioranza dei progetti per cui offriamo assistenza posseggono pochi campioni, quindi è bene disporre di un piccolo pacchetto. In precedenza, siamo stati limitati dai costi, quindi ciò potrebbe aprire la strada ad altri studi in cui osservare i geni più grandi con meno campioni." I pannelli su richiesta Ion AmpliSeq On-Demand Panels sono personalizzati dai clienti sulla base dello strumento di progettazione Ion AmpliSeq Designer (www.ampliseq.com) selezionando da un crescente archivio composto da geni bersaglio altamente ottimizzati importanti per la ricerca sulle malattie genetiche. Il database per le malattie genetiche dello strumento, che consente la selezione dei geni in funzione dell'area di ricerca delle malattie, è composto da dati ricevuti da archivi pubblici, quale il database del Medical Subject Headings (MeSH/Termini medici), e include set di primer basati sulle migliaia di progetti sperimentati che sono anche stati verificati in wet lab (laboratorio umido) per garantirne la resa. I pannelli sono quindi ordinati istantaneamente in pratici pacchetti appositi per le esigenze sperimentali e ridurre i costi iniziali. "Il sequenziamento mirato ricorrendo a pannelli personalizzati in base al tipo di cliente ha dimostrato di essere una metodologia popolare a guida della ricerca traslazionale, ma per malattie non comuni e complesse quali quelle genetiche la maggioranza dei laboratori non hanno un numero di campioni tali da giustificare un investimento importante a livello temporale ed economico", ha detto Joydeep Goswami, presidente del Clinical Next-Generation Sequencing and Oncology (Oncologia e NGS clinici) presso Thermo Fisher Scientific. "Semplificando la modalità di personalizzazione per gli utenti quanto a contenuto e pacchetto, i ricercatori clinici possono concentrarsi sui geni bersaglio interessati che orienteranno scoperte maggiori senza elevati costi iniziali e il rischio di sprechi." Thermo Fisher illustrerà il nuovo software progettuale Ion AmpliSeq Designer Software ai delegati che lo richiederanno presso l'ESHG 2017. La società ospiterà anche un workshop con colloqui di utenti nell'accesso iniziale dei pannelli su richiesta Ion AmpliSeq On-Demand Panels e altre nuove tecnologie di Thermo Fisher. Il workshop complementare, intitolato New Products to Enable Discovery of De Novo and Germline Mutations (Nuovi prodotti per la scoperta di mutazioni genetiche e de novo), avrà luogo domenica 28 maggio alle ore 11:15 CET nella sala Ballerup Room presso il Bella Center Copenhagen (BCC). Tra i presentatori del workshop ci saranno: Informazioni su Thermo Fisher Scientific Thermo Fisher Scientific Inc. è leader mondiale al servizio della scienza, con un fatturato pari a 18 miliardi di dollari e oltre 55.000 dipendenti nel mondo. La nostra missione risiede nel consentire ai nostri clienti di rendere il mondo più salubre, pulito e sicuro. Aiutiamo i nostri clienti ad accelerare la ricerca nelle scienze naturali, risolvere problematiche analitiche complesse, migliorare le diagnosi per i pazienti e aumentare la produttività dei laboratori. Tramite i nostri marchi principali – Thermo Scientific, Applied Biosystems, Invitrogen, Fisher Scientific e Unity Lab Services – offriamo una combinazione ineguagliabile di tecnologie innovative, convenienza nell'acquisto e assistenza completa. Per ulteriori informazioni visitare il sito web www.thermofisher.com.
News Article | May 10, 2017
As part of the SyncD3 webinar series and virtual event, Thermo Fisher Scientific will discuss cutting-edge tools and topics in the fields of drug discovery & development. These tolls and topics include HCS/phenotypic assays, functional genomics & drug metabolism, 3D models/organoids, diseased models, CRISPR and stem cells. In this webinar, attendees will gain a better understanding of where these areas intersect, the impact on drug discovery and development, as well as the future of the industry during a live panel discussion. They will learn from real field scientists and researchers who are working in the ADME/Tox and Drug Discovery fields. The speakers for this event will be Dr. David Piper, director of research and development of Cellular Biology at Thermo Fisher Scientific, and Dr. Mark Kennedy, Scientist at Thermo Fisher Scientific, serving in the Cell Biology ADME/Tox group. Dr. Piper earned a doctorate in neuroscience from the University of Utah. He has led teams at Thermo Fisher Scientific for more than 10 years in the development of products and services, and now as an R&D Director for the Cell Biology and Synthetic Biology businesses, he leads teams that provide molecular biology and cellular biology services. Dr. Kennedy received his Ph.D. from Memorial University. Now in his current role at Thermo Fisher Scientific, Kennedy is part of a team that focuses on the development of new in vitro 3D cell culture models. His ongoing work focusses on the utilization of human embryonic stem cells, iPSCs and primary cells, and their application in spheroids, organoids and co-culture systems. LabRoots will host the event May 16, 2017, beginning at 9:00 a.m. PDT, 12:00 p.m. EDT. To learn more about this event or to register for free, click here. ABOUT THERMO FISHER SCIENTIFIC Thermo Fisher Scientific Inc. is the world leader in serving science, with revenues of $17 billion and more than 50,000 employees in 50 countries. Our mission is to enable our customers to make the world healthier, cleaner and safer. We help our customers accelerate life sciences research, solve complex analytical challenges, improve patient diagnostics and increase laboratory productivity. Through our premier brands – Thermo Scientific, Applied Biosystems, Invitrogen, Fisher Scientific and Unity Lab Services – we offer an unmatched combination of innovative technologies, purchasing convenience and comprehensive support. For more information, please visit http://www.thermofisher.com ABOUT LABROOTS LabRoots is the leading scientific social networking website, which provides daily scientific trending news, as well as produces educational virtual events and webinars, on the latest discoveries and advancements in science. Contributing to the advancement of science through content sharing capabilities, LabRoots is a powerful advocate in amplifying global networks and communities. Founded in 2008, LabRoots emphasizes digital innovation in scientific collaboration and learning, and is a primary source for current scientific news, webinars, virtual conferences, and more. LabRoots has grown into the world’s largest series of virtual events within the Life Sciences and Clinical Diagnostics community.