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  • 99
    Thermo Fisher superscript iii cdna synthesis kit
    Isolation and Purification of Mx, ORS, DP, DF, and Mc Populations (A) Schematic of isolation procedure. After removing subcutaneous fat by dissection, and epidermis/upper follicle segment by enzymatic digestion, single-cell suspensions were prepared from pure dermis and subjected to <t>three</t> FACS schemes to purify five populations of cells: Mx, GFP low RFP − ; ORS, GFP high RFP − ; DP, RFP high GFP − CD34 − CD45 − CD117 − ; DF, RFP − GFP − CD34 − CD45 − CD117 − ; Mc, RFP high GFP − CD117 + . (B) Immunofluorescence analyses of FACS isolated cell populations. Frozen skin sections (hair bulb) and relevant cytospin populations were stained with Abs as color-coded and indicated. At the right of each set is quantification of percentage of cells that expressed the marker. Note: ~10% of DP and DF cells lysed on cytospin. and hence did not stain with any markers. β4, β4 integrin; Tyr, tyrosinase; Vim, vimentin; white line, basement membrane. (C) RT-PCR: <t>cDNA</t> fragments were resolved by agarose gel electrophoresis, and the gene detected is denoted at left. All fragments were of the expected size. Expression of Msx2, vimentin, and β4 in multiple populations was later confirmed. (D) Cell cycle differences in cell populations. Profiles of the five purified populations were performed by FACS. Anti-BrdU immunofluorescence is from a P4 backskin follicle from a mouse injected intraperitoneally with 50 μg/g 5-bromo-2′-deoxyuridine (BrdU) (Sigma-Aldrich) and analyzed 4 h later. Note greatest incorporation in Mx and ORS.
    Superscript Iii Cdna Synthesis Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2828 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher cellsdirect one step qrt pcr kit
    HTM cell characterization. ( A ) Representative phase contrast micrographs of reference, HTM01, HTM11, and HTM12 cell strains with sex/age information. Scale bar, 250 µm. ( B ) mRNA fold-change of MYOC by <t>qRT-PCR</t> at 7 d (N = 3 per group and donor; *p
    Cellsdirect One Step Qrt Pcr Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 710 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Thermo Fisher cellsdirect
    HTM cell characterization. ( A ) Representative phase contrast micrographs of reference, HTM01, HTM11, and HTM12 cell strains with sex/age information. Scale bar, 250 µm. ( B ) mRNA fold-change of MYOC by <t>qRT-PCR</t> at 7 d (N = 3 per group and donor; *p
    Cellsdirect, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 323 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher cellsdirect kit
    HTM cell characterization. ( A ) Representative phase contrast micrographs of reference, HTM01, HTM11, and HTM12 cell strains with sex/age information. Scale bar, 250 µm. ( B ) mRNA fold-change of MYOC by <t>qRT-PCR</t> at 7 d (N = 3 per group and donor; *p
    Cellsdirect Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 147 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher cellsdirect resuspension lysis buffers includes resuspension buffer lysis buffer
    HTM cell characterization. ( A ) Representative phase contrast micrographs of reference, HTM01, HTM11, and HTM12 cell strains with sex/age information. Scale bar, 250 µm. ( B ) mRNA fold-change of MYOC by <t>qRT-PCR</t> at 7 d (N = 3 per group and donor; *p
    Cellsdirect Resuspension Lysis Buffers Includes Resuspension Buffer Lysis Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher cellsdirect reaction mix
    HTM cell characterization. ( A ) Representative phase contrast micrographs of reference, HTM01, HTM11, and HTM12 cell strains with sex/age information. Scale bar, 250 µm. ( B ) mRNA fold-change of MYOC by <t>qRT-PCR</t> at 7 d (N = 3 per group and donor; *p
    Cellsdirect Reaction Mix, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 130 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Isolation and Purification of Mx, ORS, DP, DF, and Mc Populations (A) Schematic of isolation procedure. After removing subcutaneous fat by dissection, and epidermis/upper follicle segment by enzymatic digestion, single-cell suspensions were prepared from pure dermis and subjected to three FACS schemes to purify five populations of cells: Mx, GFP low RFP − ; ORS, GFP high RFP − ; DP, RFP high GFP − CD34 − CD45 − CD117 − ; DF, RFP − GFP − CD34 − CD45 − CD117 − ; Mc, RFP high GFP − CD117 + . (B) Immunofluorescence analyses of FACS isolated cell populations. Frozen skin sections (hair bulb) and relevant cytospin populations were stained with Abs as color-coded and indicated. At the right of each set is quantification of percentage of cells that expressed the marker. Note: ~10% of DP and DF cells lysed on cytospin. and hence did not stain with any markers. β4, β4 integrin; Tyr, tyrosinase; Vim, vimentin; white line, basement membrane. (C) RT-PCR: cDNA fragments were resolved by agarose gel electrophoresis, and the gene detected is denoted at left. All fragments were of the expected size. Expression of Msx2, vimentin, and β4 in multiple populations was later confirmed. (D) Cell cycle differences in cell populations. Profiles of the five purified populations were performed by FACS. Anti-BrdU immunofluorescence is from a P4 backskin follicle from a mouse injected intraperitoneally with 50 μg/g 5-bromo-2′-deoxyuridine (BrdU) (Sigma-Aldrich) and analyzed 4 h later. Note greatest incorporation in Mx and ORS.

    Journal: PLoS Biology

    Article Title: Molecular Dissection of Mesenchymal-Epithelial Interactions in the Hair FollicleMolecular Signatures of the Developing Hair Follicle

    doi: 10.1371/journal.pbio.0030331

    Figure Lengend Snippet: Isolation and Purification of Mx, ORS, DP, DF, and Mc Populations (A) Schematic of isolation procedure. After removing subcutaneous fat by dissection, and epidermis/upper follicle segment by enzymatic digestion, single-cell suspensions were prepared from pure dermis and subjected to three FACS schemes to purify five populations of cells: Mx, GFP low RFP − ; ORS, GFP high RFP − ; DP, RFP high GFP − CD34 − CD45 − CD117 − ; DF, RFP − GFP − CD34 − CD45 − CD117 − ; Mc, RFP high GFP − CD117 + . (B) Immunofluorescence analyses of FACS isolated cell populations. Frozen skin sections (hair bulb) and relevant cytospin populations were stained with Abs as color-coded and indicated. At the right of each set is quantification of percentage of cells that expressed the marker. Note: ~10% of DP and DF cells lysed on cytospin. and hence did not stain with any markers. β4, β4 integrin; Tyr, tyrosinase; Vim, vimentin; white line, basement membrane. (C) RT-PCR: cDNA fragments were resolved by agarose gel electrophoresis, and the gene detected is denoted at left. All fragments were of the expected size. Expression of Msx2, vimentin, and β4 in multiple populations was later confirmed. (D) Cell cycle differences in cell populations. Profiles of the five purified populations were performed by FACS. Anti-BrdU immunofluorescence is from a P4 backskin follicle from a mouse injected intraperitoneally with 50 μg/g 5-bromo-2′-deoxyuridine (BrdU) (Sigma-Aldrich) and analyzed 4 h later. Note greatest incorporation in Mx and ORS.

    Article Snippet: Quality was assessed by RNA 6000 Pico Assay (Agilent Technologies, Palo Alto, California, United States), and 800 ng were primed with oligo(dT)-T7 primer and reverse transcribed (Superscript III cDNA synthesis kit; Invitrogen, Carlsbad, California, United States).

    Techniques: Isolation, Purification, Dissection, FACS, Immunofluorescence, Staining, Marker, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Expressing, Injection

    Implementation of Array Anal yses to Examine Characteristics and Dynamics of the Follicle DP Niche (A) Semi-quantitative RT-PCR on mRNAs isolated from each population. Shown are representative data from molecular signature genes (see Figure 4 ) whose expression patterns in the DP niche environment had been previously uncharacterized. In this case, categories were consolidated into three groups: Signal transduction, transcription/nuclear, and cytoskeleton/ECM/cell adhesion. For each primer set, at least three different cycles were employed, and the resulting cDNA fragments were resolved by agarose gel electrophoresis along with DNA size markers to confirm that bands were of the expected sizes. For each gene, the data presented were from the cycle that provided the most meaningful comparisons. Note: bands seen in > 1 fraction accurately reflect mRNA expression at the differences in levels shown. (B) Immunohistochemistry and in situ hybridizations. Skin sections were taken from 2-mo-old K14-GFPactin mice [ 49 ] whose follicles were at the transition from the resting to growing (telogen to anagen) stage of the hair cycle (8 wk) or from P4 WT mice (full anagen follicles) (all others). Sections were subjected to either immunofluorescence using color-coded Abs as indicated or in situ hybridization using the indicated biotinylated cRNA probes (sense controls were negative).

    Journal: PLoS Biology

    Article Title: Molecular Dissection of Mesenchymal-Epithelial Interactions in the Hair FollicleMolecular Signatures of the Developing Hair Follicle

    doi: 10.1371/journal.pbio.0030331

    Figure Lengend Snippet: Implementation of Array Anal yses to Examine Characteristics and Dynamics of the Follicle DP Niche (A) Semi-quantitative RT-PCR on mRNAs isolated from each population. Shown are representative data from molecular signature genes (see Figure 4 ) whose expression patterns in the DP niche environment had been previously uncharacterized. In this case, categories were consolidated into three groups: Signal transduction, transcription/nuclear, and cytoskeleton/ECM/cell adhesion. For each primer set, at least three different cycles were employed, and the resulting cDNA fragments were resolved by agarose gel electrophoresis along with DNA size markers to confirm that bands were of the expected sizes. For each gene, the data presented were from the cycle that provided the most meaningful comparisons. Note: bands seen in > 1 fraction accurately reflect mRNA expression at the differences in levels shown. (B) Immunohistochemistry and in situ hybridizations. Skin sections were taken from 2-mo-old K14-GFPactin mice [ 49 ] whose follicles were at the transition from the resting to growing (telogen to anagen) stage of the hair cycle (8 wk) or from P4 WT mice (full anagen follicles) (all others). Sections were subjected to either immunofluorescence using color-coded Abs as indicated or in situ hybridization using the indicated biotinylated cRNA probes (sense controls were negative).

    Article Snippet: Quality was assessed by RNA 6000 Pico Assay (Agilent Technologies, Palo Alto, California, United States), and 800 ng were primed with oligo(dT)-T7 primer and reverse transcribed (Superscript III cDNA synthesis kit; Invitrogen, Carlsbad, California, United States).

    Techniques: Quantitative RT-PCR, Isolation, Expressing, Transduction, Agarose Gel Electrophoresis, Immunohistochemistry, In Situ, Mouse Assay, Immunofluorescence, In Situ Hybridization

    Analysis of inflammatory gene and protein expression in tongue tissues.A. Two chemokines (CXCL1, CXCL2), a neutrophil-specific antigen (CD177) and an epithelial cell-derived neutrophil-activating cytokine (IL-17C) were tested in the same RNA samples surveyed by microarray analysis on day 5 post infection. After cDNA synthesis, equal amounts from three mice per group were mixed and analysed in triplicate by RT-qPCR. All genes were significantly ( P

    Journal: Cellular Microbiology

    Article Title: Streptococcal co-infection augments Candida pathogenicity by amplifying the mucosal inflammatory response

    doi: 10.1111/cmi.12216

    Figure Lengend Snippet: Analysis of inflammatory gene and protein expression in tongue tissues.A. Two chemokines (CXCL1, CXCL2), a neutrophil-specific antigen (CD177) and an epithelial cell-derived neutrophil-activating cytokine (IL-17C) were tested in the same RNA samples surveyed by microarray analysis on day 5 post infection. After cDNA synthesis, equal amounts from three mice per group were mixed and analysed in triplicate by RT-qPCR. All genes were significantly ( P

    Article Snippet: RNA concentrations and quality were determined by measuring the absorbance at 260 nm and 280 nm using the NanoDrop device. cDNA was synthesized by using SuperScript III CellsDirect cDNA Synthesis Kit® (Invitrogen), according to the manual.

    Techniques: Expressing, Derivative Assay, Microarray, Infection, Mouse Assay, Quantitative RT-PCR

    E-cadherin is downregulated in migratory PGCs. (A) Relative amount of E-cadherin ( cdh1 ) in PGCs and somatic endodermal cells (Som) isolated from stage 28–30 embryos (migratory PGCs) normalized to E-cadherin level in the corresponding cell type isolated from stage 17–19 embryos (pre-migratory PGCs) measured by quantitative RT PCR. Relative amount was calculated by ΔΔCt method (see Material and Methods) using three independent cDNA preparations. Error bars represent standard deviation. * corresponds to P

    Journal: Biology Open

    Article Title: Migratory and adhesive properties of Xenopus laevis primordial germ cells in vitro

    doi: 10.1242/bio.20135140

    Figure Lengend Snippet: E-cadherin is downregulated in migratory PGCs. (A) Relative amount of E-cadherin ( cdh1 ) in PGCs and somatic endodermal cells (Som) isolated from stage 28–30 embryos (migratory PGCs) normalized to E-cadherin level in the corresponding cell type isolated from stage 17–19 embryos (pre-migratory PGCs) measured by quantitative RT PCR. Relative amount was calculated by ΔΔCt method (see Material and Methods) using three independent cDNA preparations. Error bars represent standard deviation. * corresponds to P

    Article Snippet: 30 cells from each cell population were used for the preparation of cDNA using SuperScript III CellsDirect cDNA Synthesis System (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol.

    Techniques: Isolation, Quantitative RT-PCR, Standard Deviation

    Detection of LASV clade II and IV. a Schematic of LASV SHERLOCK assays targeting the two most common clades of LASV: clades II (LASV-II assay) and IV (LASV-IV assay). For the LASV-II assay, three crRNAs were designed and tested. Two crRNAs are multiplexed to encompass the clade’s genetic diversity (IIA/IIB or IIA/IIC). Each crRNA was tested using three technical replicates. b – d Heat maps are measured in fluorescence (a.u.). b Detection of LASV RNA from suspected LF clinical samples using crRNAs IIA, IIB, IIC, or a combination of crRNAs. c Test of cross-reactivity between different viral species using MARV, EBOV, and LASV viral seedstock cDNA. The LASV-II and LASV-IV assays do not cross-react with MARV or EBOV seed stocks. d Test of cross-reactivity between LASV clade-specific assays using clinical samples from recent outbreaks in Nigeria and Sierra Leone. The LASV-II and LASV-IV assays provide clade-specific detection. e SHERLOCK testing using the LASV-II assay of RNA extracted from seven confirmed LASV-positive and three confirmed LASV-negative samples collected from suspected LF patients in Nigeria during the 2018 outbreak. Error bar indicates 95% confidence interval. f Results from e were compared head-to-head to those from the gold standard Nikisins RT-qPCR assay, next-generation sequencing (genome assembled), and lateral flow detection. g SHERLOCK testing using the LASV-IV assay of RNA extracted from seven confirmed LASV-positive and three confirmed LASV-negative samples collected from suspected LF patients in Sierra Leone. Error bar indicates 95% confidence interval. h Results from g were compared head-to-head to those from the gold standard Nikisins RT-qPCR assay, a second Broad RT-qPCR, NGS, and lateral flow detection. Source dare are in the Source Data file.

    Journal: Nature Communications

    Article Title: Deployable CRISPR-Cas13a diagnostic tools to detect and report Ebola and Lassa virus cases in real-time

    doi: 10.1038/s41467-020-17994-9

    Figure Lengend Snippet: Detection of LASV clade II and IV. a Schematic of LASV SHERLOCK assays targeting the two most common clades of LASV: clades II (LASV-II assay) and IV (LASV-IV assay). For the LASV-II assay, three crRNAs were designed and tested. Two crRNAs are multiplexed to encompass the clade’s genetic diversity (IIA/IIB or IIA/IIC). Each crRNA was tested using three technical replicates. b – d Heat maps are measured in fluorescence (a.u.). b Detection of LASV RNA from suspected LF clinical samples using crRNAs IIA, IIB, IIC, or a combination of crRNAs. c Test of cross-reactivity between different viral species using MARV, EBOV, and LASV viral seedstock cDNA. The LASV-II and LASV-IV assays do not cross-react with MARV or EBOV seed stocks. d Test of cross-reactivity between LASV clade-specific assays using clinical samples from recent outbreaks in Nigeria and Sierra Leone. The LASV-II and LASV-IV assays provide clade-specific detection. e SHERLOCK testing using the LASV-II assay of RNA extracted from seven confirmed LASV-positive and three confirmed LASV-negative samples collected from suspected LF patients in Nigeria during the 2018 outbreak. Error bar indicates 95% confidence interval. f Results from e were compared head-to-head to those from the gold standard Nikisins RT-qPCR assay, next-generation sequencing (genome assembled), and lateral flow detection. g SHERLOCK testing using the LASV-IV assay of RNA extracted from seven confirmed LASV-positive and three confirmed LASV-negative samples collected from suspected LF patients in Sierra Leone. Error bar indicates 95% confidence interval. h Results from g were compared head-to-head to those from the gold standard Nikisins RT-qPCR assay, a second Broad RT-qPCR, NGS, and lateral flow detection. Source dare are in the Source Data file.

    Article Snippet: AMPure RNA clean beads (Beckman Coulter Genomics) were used to clean and concentrate samples. cDNA synthesis was performed using the Superscript III kit (Thermo Fischer) plus dNTPs, random primers, and SUPERASE-IN for first-strand synthesis.

    Techniques: Ii Assay, Fluorescence, Quantitative RT-PCR, Next-Generation Sequencing

    Detection of EBOV. a Schematic of the SHERLOCK EBOV assay. b , c Detection of a serial dilution of EBOV synthetic DNA using ( b ) mean fluorescence of three technical replicates and ( c ) lateral flow readouts. Error bars indicate ±1 SD for three technical replicates. d Test of cross-reactivity using MARV, EBOV, and LASV viral seedstock cDNA. Heat map is measured in Fluorescence (a.u.). e SHERLOCK testing of cDNA extracted from 12 confirmed EBOV-positive and 4 confirmed EBOV-negative samples collected from suspected EVD patients during the 2014 outbreak in Sierra Leone. Error bars indicate 95% confidence interval. f Four of the samples from e were also tested by collaborators using lateral flow detection. g , h Detection of serial dilution of synthetic RNA from Ituri, DRC and Makona, Sierra Leone using ( g ) fluorescence where error bars indicate ±1 SD for three technical replicates and ( h ) lateral flow readouts carried out at USAMRIID. Source data are in the Source Data file.

    Journal: Nature Communications

    Article Title: Deployable CRISPR-Cas13a diagnostic tools to detect and report Ebola and Lassa virus cases in real-time

    doi: 10.1038/s41467-020-17994-9

    Figure Lengend Snippet: Detection of EBOV. a Schematic of the SHERLOCK EBOV assay. b , c Detection of a serial dilution of EBOV synthetic DNA using ( b ) mean fluorescence of three technical replicates and ( c ) lateral flow readouts. Error bars indicate ±1 SD for three technical replicates. d Test of cross-reactivity using MARV, EBOV, and LASV viral seedstock cDNA. Heat map is measured in Fluorescence (a.u.). e SHERLOCK testing of cDNA extracted from 12 confirmed EBOV-positive and 4 confirmed EBOV-negative samples collected from suspected EVD patients during the 2014 outbreak in Sierra Leone. Error bars indicate 95% confidence interval. f Four of the samples from e were also tested by collaborators using lateral flow detection. g , h Detection of serial dilution of synthetic RNA from Ituri, DRC and Makona, Sierra Leone using ( g ) fluorescence where error bars indicate ±1 SD for three technical replicates and ( h ) lateral flow readouts carried out at USAMRIID. Source data are in the Source Data file.

    Article Snippet: AMPure RNA clean beads (Beckman Coulter Genomics) were used to clean and concentrate samples. cDNA synthesis was performed using the Superscript III kit (Thermo Fischer) plus dNTPs, random primers, and SUPERASE-IN for first-strand synthesis.

    Techniques: Serial Dilution, Fluorescence

    HTM cell characterization. ( A ) Representative phase contrast micrographs of reference, HTM01, HTM11, and HTM12 cell strains with sex/age information. Scale bar, 250 µm. ( B ) mRNA fold-change of MYOC by qRT-PCR at 7 d (N = 3 per group and donor; *p

    Journal: bioRxiv

    Article Title: A tissue-engineered human trabecular meshwork hydrogel for advanced glaucoma disease modeling

    doi: 10.1101/2020.07.31.229229

    Figure Lengend Snippet: HTM cell characterization. ( A ) Representative phase contrast micrographs of reference, HTM01, HTM11, and HTM12 cell strains with sex/age information. Scale bar, 250 µm. ( B ) mRNA fold-change of MYOC by qRT-PCR at 7 d (N = 3 per group and donor; *p

    Article Snippet: The monolayer HTM cells were processed for quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and immunocytochemistry (ICC) analyses.

    Techniques: Quantitative RT-PCR

    Confirmation of genes in SMEP by qRT-PCR. A heat map represents the expression levels for 29 selected genes using qRT-PCR from 151 SST-REX identified genes and 81 potential NSC SMEP by microarray. Each colored grid in the heat map represents the relative abundance of the transcript compared to the Gapdh level. Each gene was identified by either single or multiple niche cell type SMEP (parentheses on the left column). N: NSCs, T: TAPs, A: astrocytes, Ep: ependymal cells, C: choroid plexus, En: endothelial cells. * indicates the SMEP from microarray experiment.

    Journal: PLoS ONE

    Article Title: The Molecular Profiles of Neural Stem Cell Niche in the Adult Subventricular Zone

    doi: 10.1371/journal.pone.0050501

    Figure Lengend Snippet: Confirmation of genes in SMEP by qRT-PCR. A heat map represents the expression levels for 29 selected genes using qRT-PCR from 151 SST-REX identified genes and 81 potential NSC SMEP by microarray. Each colored grid in the heat map represents the relative abundance of the transcript compared to the Gapdh level. Each gene was identified by either single or multiple niche cell type SMEP (parentheses on the left column). N: NSCs, T: TAPs, A: astrocytes, Ep: ependymal cells, C: choroid plexus, En: endothelial cells. * indicates the SMEP from microarray experiment.

    Article Snippet: Each frozen sample was thawed and added 4 µl of a solution containing a mixture of RT/Taq enzyme (CellsDirect qRT-PCR kit, Invitrogen), combined primers, and nuclease-free water.

    Techniques: Quantitative RT-PCR, Expressing, Microarray

    IL-6 paracrine loop ( A ) Pro-tumorigenic inflammatory environment during CML development. Changes in the indicated growth factors and interleukin levels were determined using an antibody-based protein array on pools of sera from 16 Cnt and 7 BA mice 6 weeks after doxycycline withdrawal. Results are expressed as fold change relative to levels measured in Cnt mice (set to 1). ( B ) Modulation of serum IL-6 levels. ELISA measurement of IL-6 concentration in serum of individual Cnt and BA primary mice (left graphs), and mice transplanted with the indicated populations (right graphs). Results are expressed as mean ± SEM (n=2-6). ND stands for “not detectable”. (C) qRT-PCR analysis of non-hematopoietic BM cells (stroma), CD48, MPP, CMP, GMP, mature myeloid and B cells for Il-6 mRNA levels. Results are expressed as mean ± SEM (n=3). (D) Representative FACS histograms showing expression of the α-chain of IL-6 receptor on the indicated Cnt (black) and BA + (red) populations. Dashed lines show isotype control negative boundaries. Data are representative of two independent experiments. ( E ) Effect of IL-6 on lymphoid differentiation from BA + MPPs. The FACS plots show a representative phenotypic analysis of CD19 (lymphoid) and Mac-1 (myeloid) markers after 8 days of in vitro culture (n=2). ( F ) Differential effect of IL-6 on myeloid differentiation from BA + MPPs and GMPs. The graph on the left show the fold changes in colony numbers when comparing conditions with or without IL-6 (set to 1). Results are expressed as mean ± SEM (n=2-3; **p≤5×10 -4 ). The photographs on the right show representative myeloid colonies generated in both conditions from BA + MPPs after 10 days of in vitro culture. ( G ) Model describing the IL-6 paracrine loop in CML development identified in this study.

    Journal: Cancer cell

    Article Title: IL-6 controls leukemic multipotent progenitor cell fate and contributes to chronic myelogenous leukemia development

    doi: 10.1016/j.ccr.2011.10.012

    Figure Lengend Snippet: IL-6 paracrine loop ( A ) Pro-tumorigenic inflammatory environment during CML development. Changes in the indicated growth factors and interleukin levels were determined using an antibody-based protein array on pools of sera from 16 Cnt and 7 BA mice 6 weeks after doxycycline withdrawal. Results are expressed as fold change relative to levels measured in Cnt mice (set to 1). ( B ) Modulation of serum IL-6 levels. ELISA measurement of IL-6 concentration in serum of individual Cnt and BA primary mice (left graphs), and mice transplanted with the indicated populations (right graphs). Results are expressed as mean ± SEM (n=2-6). ND stands for “not detectable”. (C) qRT-PCR analysis of non-hematopoietic BM cells (stroma), CD48, MPP, CMP, GMP, mature myeloid and B cells for Il-6 mRNA levels. Results are expressed as mean ± SEM (n=3). (D) Representative FACS histograms showing expression of the α-chain of IL-6 receptor on the indicated Cnt (black) and BA + (red) populations. Dashed lines show isotype control negative boundaries. Data are representative of two independent experiments. ( E ) Effect of IL-6 on lymphoid differentiation from BA + MPPs. The FACS plots show a representative phenotypic analysis of CD19 (lymphoid) and Mac-1 (myeloid) markers after 8 days of in vitro culture (n=2). ( F ) Differential effect of IL-6 on myeloid differentiation from BA + MPPs and GMPs. The graph on the left show the fold changes in colony numbers when comparing conditions with or without IL-6 (set to 1). Results are expressed as mean ± SEM (n=2-3; **p≤5×10 -4 ). The photographs on the right show representative myeloid colonies generated in both conditions from BA + MPPs after 10 days of in vitro culture. ( G ) Model describing the IL-6 paracrine loop in CML development identified in this study.

    Article Snippet: 100 cells were directly sorted in 5 μl of resuspension buffer from Cellsdirect™ One-Step qRT-PCR kits (Invitrogen).

    Techniques: Protein Array, Mouse Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay, Quantitative RT-PCR, FACS, Expressing, In Vitro, Generated

    Heterogeneous expression profiles of EMT-related and other genes among CTCs. RNA from CTCs was subjected to microfluidics-based single-cell qRT-PCR analysis using a BioMark HD system. Gene expression for each gene was obtained as described in materials

    Journal: The Prostate

    Article Title: Single-cell Analysis of Circulating Tumor Cells Identifies Cumulative Expression Patterns of EMT-related Genes in Metastatic Prostate Cancer

    doi: 10.1002/pros.22625

    Figure Lengend Snippet: Heterogeneous expression profiles of EMT-related and other genes among CTCs. RNA from CTCs was subjected to microfluidics-based single-cell qRT-PCR analysis using a BioMark HD system. Gene expression for each gene was obtained as described in materials

    Article Snippet: Single-cell microfluidics-based RT-PCR analysis was carried out using CellsDirectTM one-step qRT-PCR kit (cat # 11753-100, Invitrogen, Carlsbad, CA) and a microfluidics device, BioMark HD MX/HX system (cat # BMKHD-PKG-MH, Fluidigm, Inc., South San Francisco, CA) [ ].

    Techniques: Expressing, Quantitative RT-PCR

    Functional and etiological analysis of transcriptional differences between in vitro motor neuron types. (A) Fas gene expression relative to EMB MN, from the RNA-sequencing data. Data are mean±s.d., two biological replicates were analyzed per cell type. (B) Survival of MNs with or without the addition of 10 μg/ml agonistic anti-Fas antibody for 4 days. Anti-Fas antibody was added fresh to the cultures at days 0 and 2 of survival analysis. Data are mean±s.d., n =2 (EMB MN), n =4 (ESC MN) and n =3 (iMN). (C) Differential gene expression analysis between MNs. Expression is shown as log 2 fold change relative to MEF1. Genes within each MN category are color coded according to their functional roles in MN biology. Two biological replicates were analyzed per sample type. (D) Full Hox code gene expression of MNs, MEF1 and ESCs relative to iPSCs. Two biological replicates were analyzed per cell type. (E) Gene expression relative to MEF1 derived from RNA-seq data and positional information of selected Hox genes. Data are mean±s.d. Two biological replicates were analyzed per cell type. (F) qRT-PCR analyses showing relative expression of Hoxc6 and Hoxc9 in mixed MEFs (derived from whole E13.5 embryos) or posterior MEFs (derived from posterior half of E13.5 embryos) or iMNs derived from these MEF populations. Each MEF group was derived from five individual embryos per replicate (a mean of three biological replicates±s.e.m.). Two-tailed t -test, unpaired. * P

    Journal: Development (Cambridge, England)

    Article Title: Comparative genomic analysis of embryonic, lineage-converted and stem cell-derived motor neurons

    doi: 10.1242/dev.168617

    Figure Lengend Snippet: Functional and etiological analysis of transcriptional differences between in vitro motor neuron types. (A) Fas gene expression relative to EMB MN, from the RNA-sequencing data. Data are mean±s.d., two biological replicates were analyzed per cell type. (B) Survival of MNs with or without the addition of 10 μg/ml agonistic anti-Fas antibody for 4 days. Anti-Fas antibody was added fresh to the cultures at days 0 and 2 of survival analysis. Data are mean±s.d., n =2 (EMB MN), n =4 (ESC MN) and n =3 (iMN). (C) Differential gene expression analysis between MNs. Expression is shown as log 2 fold change relative to MEF1. Genes within each MN category are color coded according to their functional roles in MN biology. Two biological replicates were analyzed per sample type. (D) Full Hox code gene expression of MNs, MEF1 and ESCs relative to iPSCs. Two biological replicates were analyzed per cell type. (E) Gene expression relative to MEF1 derived from RNA-seq data and positional information of selected Hox genes. Data are mean±s.d. Two biological replicates were analyzed per cell type. (F) qRT-PCR analyses showing relative expression of Hoxc6 and Hoxc9 in mixed MEFs (derived from whole E13.5 embryos) or posterior MEFs (derived from posterior half of E13.5 embryos) or iMNs derived from these MEF populations. Each MEF group was derived from five individual embryos per replicate (a mean of three biological replicates±s.e.m.). Two-tailed t -test, unpaired. * P

    Article Snippet: Cells were collected directly into 5 μl of CellsDirect 2× Buffer (Cells Direct One-step qRT-PCR kit, Thermo).

    Techniques: Functional Assay, In Vitro, Expressing, RNA Sequencing Assay, Derivative Assay, Quantitative RT-PCR, Two Tailed Test