polymerase chain reaction pcr buffer  (Thermo Fisher)


Bioz Verified Symbol Thermo Fisher is a verified supplier  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Name:
    Buffer B 10X
    Description:
    Thermo Scientific 10X Buffer B ensures the optimum reaction conditions for specific restriction enzymes and is premixed with BSA for enhanced stability It is part of Thermo Scientific Five Buffer System which ensures the optimum reaction conditions for each restriction enzyme The system consists of 10X B blue G green O orange R red and Tango yellow buffers All restriction enzymes are supplied in color coded tubes to indicate the recommended reaction buffer The recommended buffer and or the universal Tango buffer are supplied with each enzyme To ensure consistent enzyme performance Thermo Scientific restriction enzyme buffers contain BSA which enhances the stability of many enzymes and binds contaminants that may be present in DNA preparations Multiple freeze thaw cycles of the buffers will not cause BSA precipitation Thermo Scientific restriction enzymes exhibit 100 of their certified activity in the recommended buffer However some enzymes require additives to achieve 100 activity For example AjuI AlfI BdaI BplI BseMII FaqI Eco57I Eco57MI Hin4I and TsoI require S adenosylmethionine which is supplied with the enzyme AarI and BveI require an oligonucleotide also supplied with the enzyme and Esp3I requires DTT
    Catalog Number:
    bb5
    Price:
    None
    Applications:
    Cloning|Restriction Enzyme Cloning
    Category:
    Lab Reagents and Chemicals
    Buy from Supplier


    Structured Review

    Thermo Fisher polymerase chain reaction pcr buffer
    Thermo Scientific 10X Buffer B ensures the optimum reaction conditions for specific restriction enzymes and is premixed with BSA for enhanced stability It is part of Thermo Scientific Five Buffer System which ensures the optimum reaction conditions for each restriction enzyme The system consists of 10X B blue G green O orange R red and Tango yellow buffers All restriction enzymes are supplied in color coded tubes to indicate the recommended reaction buffer The recommended buffer and or the universal Tango buffer are supplied with each enzyme To ensure consistent enzyme performance Thermo Scientific restriction enzyme buffers contain BSA which enhances the stability of many enzymes and binds contaminants that may be present in DNA preparations Multiple freeze thaw cycles of the buffers will not cause BSA precipitation Thermo Scientific restriction enzymes exhibit 100 of their certified activity in the recommended buffer However some enzymes require additives to achieve 100 activity For example AjuI AlfI BdaI BplI BseMII FaqI Eco57I Eco57MI Hin4I and TsoI require S adenosylmethionine which is supplied with the enzyme AarI and BveI require an oligonucleotide also supplied with the enzyme and Esp3I requires DTT
    https://www.bioz.com/result/polymerase chain reaction pcr buffer/product/Thermo Fisher
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    polymerase chain reaction pcr buffer - by Bioz Stars, 2020-07
    99/100 stars

    Images

    Related Articles

    Polymerase Chain Reaction:

    Article Title: Rapid analysis of heterogeneously methylated DNA using digital methylation-sensitive high resolution melting: application to the CDKN2B (p15) gene
    Article Snippet: .. PCR was performed in 100 μl PCR tubes (Corbett Research) with a final volume of 20 μl, containing 200 nmol/l of each primer, 200 μmol/l of each dNTP, 0.5 U of HotStarTaq DNA polymerase (Qiagen) in the supplied PCR buffer containing 2.5 mmol/l MgCl2 , 5 μmol/l SYTO9 (Invitrogen, Carlsbad, CA), and 10 ng of bisulphite-treated DNA. .. The initial denaturation (95°C, 15 minutes) was followed by 50 cycles for MS-HRM of 10 seconds at 95°C, 30 seconds at 59°C, 30 seconds at 72°C; one cycle of 1 minute at 95°C, 72°C for 1.5 minutes and a HRM step from 65°C to 90°C rising at 0.2°C per second, and holding for 1 second after each stepwise increment.

    Article Title: miR-138 overexpression is more powerful than hTERT knockdown to potentiate apigenin for apoptosis in neuroblastoma in vitro and in vivo
    Article Snippet: .. Briefly, PCR reaction was performed after mixing 0.5 μl of miR-138 specific primers (10 μM), 2.5 μl of PCR buffer (10μl), 0.75 μl of MgCl2 (50 mM), 0.5 μl of dNTPs (10 mM, Invitrogen, Carlsbad, CA, USA), 0.2 μl of Taq Platinum DNA polymerase (5 U/μl, Invitrogen, Carlsbad, CA, USA) and 2 μl of RT product in a PCR thermal cycler (Eppendorf mastercycler gradient). .. The cycling protocol consisted of an initial 10 min denaturation step at 95°C, followed by 35 cycles of PCR amplification of the transcripts using a program (denaturation at 95°C for 15 s, and annealing at 52 °C for 30 s, and 72°C extension for 30 s), and final extension at 72°C for 7 min.

    Article Title: The mRNA of L-Type Calcium Channel Elevated in Colon Cancer
    Article Snippet: .. Each PCR reaction of 50 μl was composed of 1 μl of cDNA made from 195 ng of total RNA (group A samples) or 47 ng (group B samples), 1× PCR buffer, pH 8.5, with a 1.5 or 2.5 mmol/L Mg2+ Hot Wax Bead (Invitrogen, San Diego, CA), 0.2 mmol/L dATP, dCTP, dGTP, dTTP, 2.5 units of Taq DNA Polymerase (Life Technologies) and 0.5 μmol/L primers, except the β-actin primers which were used at 0.1 μmol/L. .. The DNA Thermal Cycler 480 (Perkin-Elmer, Norwalk, CT) was programmed for 30 (VDR, β-actin), 34 (HGF), and 35 (α1C , CK20) cycles.

    Article Title: Molecular profile of oligodendrogliomas in young patients
    Article Snippet: .. PCR amplification was performed in a total of 10 µL of reaction mixture containing 50 ng of tumor DNA, 1 µL of 10× PCR buffer, 0.8 µL of 10 mM dNTPs, 0.25 µL of each forward and reverse primers, and 0.2 µL of AmpliTaq Gold PCR Master Mix (Applied Biosystems). .. The reaction mixture was subjected to an initial denaturation of 95°C for 5 min, followed by 35 cycles of amplification consisting of denaturation at 95°C for 1 min, annealing at 57°C for 45 s, and extension 72°C for 2 min. Bidirectional sequencing was performed using ABI 3730 sequencer (Applied Biosystems).

    Article Title: Data in support of the detection of genetically modified organisms (GMOs) in food and feed samples
    Article Snippet: .. The final concentrations of each PCR were as follows: l× of 10× PCR buffer (fermentase); 100 ng of genomic DNA; 0.4 pm of each primers; 0.32 mM of dNTPs mix; 2 mM MgCl2 ; 0.5 unit/reaction of (Fermentas) Taq DNA polymerase. ..

    Article Title: Molecular Characterization of Porcine MMP19 and MMP23B Genes and Its Association with Immune Traits
    Article Snippet: .. The PCR 50 µL reaction consisted of 100 ng DNA or cDNA template, 5 µL 10x PCR buffer, 4 µL 2.5 mM dNTP mix, 1 µL sense primer (100 ng/µL), 1 µL antisense primer (100 ng/µL), 0.5 µL 5 U r-Taq DNA polymerase (Fermentas, Vilnius, Lithuania) and sterile water. .. The PCR amplification was initiated with a 5-minute denaturation at 95°C and continued with the following parameters (35 cycles): 30 sec at 95°C (denaturation), 30 sec at Tm specific for primer (annealing) and 30 sec at 72°C (elongation), followed by 5 min at 72°C (elongation), and stored at 4°C until analysis.

    Article Title: Identification of a Novel Methylated Gene in Nasopharyngeal Carcinoma: TTC40
    Article Snippet: .. Briefly, 1 μ g of DNA in 40 μ L was digested with either 10 units of methylation-sensitive restriction enzyme Hpa II (Fermentas) or 10 units of methylation-insensitive isoschizomer Msp I (Fermentas) at 37°C for 16 h. Aliquot of restriction-digested DNA (4 μ L) was amplified in a final volume of 50 μ L containing 1x PCR buffer, 50 pmol of a single primer MLG2 (5′-AACCCTCACCCTAACCCCGG-3′), 200 μ M of each dNTP, 2 mM of MgCl2 , and 1.25 units of Taq DNA polymerase (Fermentas). .. The reactions were carried out in a thermal cycler (Applied Biosystems 2720) for five cycles under low stringency (94°C for 30 s, 40°C for 60 s, and 72°C for 90 s) followed by 30 cycles under high stringency (95°C for 15 s, 55°C for 15 s, and 72°C for 60 s).

    Article Title: Expression of an Altered Ribonucleotide Reductase Activity Associated with the Replication of Murine Cytomegalovirus in Quiescent Fibroblasts
    Article Snippet: .. A 2-μg quantity of RNA was retrotranscribed at 42°C for 60 min in PCR buffer (1.5 mM MgCl2 ) containing 5 μM random primers, 0.5 mM each dNTP, and 100 U of Moloney murine leukemia virus reverse transcriptase (Ambion) in a final volume of 20 μl. .. The resulting cDNAs were amplified with the following primers for MCMV M45: upstream primer, 5′ ATG GCT CGC ATC CGC CGC TAC-3′; downstream primer, 5′ GGC CGA GTA GAA CTG AGC GCG-3′.

    Amplification:

    Article Title: Molecular profile of oligodendrogliomas in young patients
    Article Snippet: .. PCR amplification was performed in a total of 10 µL of reaction mixture containing 50 ng of tumor DNA, 1 µL of 10× PCR buffer, 0.8 µL of 10 mM dNTPs, 0.25 µL of each forward and reverse primers, and 0.2 µL of AmpliTaq Gold PCR Master Mix (Applied Biosystems). .. The reaction mixture was subjected to an initial denaturation of 95°C for 5 min, followed by 35 cycles of amplification consisting of denaturation at 95°C for 1 min, annealing at 57°C for 45 s, and extension 72°C for 2 min. Bidirectional sequencing was performed using ABI 3730 sequencer (Applied Biosystems).

    Article Title: Identification of a Novel Methylated Gene in Nasopharyngeal Carcinoma: TTC40
    Article Snippet: .. Briefly, 1 μ g of DNA in 40 μ L was digested with either 10 units of methylation-sensitive restriction enzyme Hpa II (Fermentas) or 10 units of methylation-insensitive isoschizomer Msp I (Fermentas) at 37°C for 16 h. Aliquot of restriction-digested DNA (4 μ L) was amplified in a final volume of 50 μ L containing 1x PCR buffer, 50 pmol of a single primer MLG2 (5′-AACCCTCACCCTAACCCCGG-3′), 200 μ M of each dNTP, 2 mM of MgCl2 , and 1.25 units of Taq DNA polymerase (Fermentas). .. The reactions were carried out in a thermal cycler (Applied Biosystems 2720) for five cycles under low stringency (94°C for 30 s, 40°C for 60 s, and 72°C for 90 s) followed by 30 cycles under high stringency (95°C for 15 s, 55°C for 15 s, and 72°C for 60 s).

    Methylation:

    Article Title: Identification of a Novel Methylated Gene in Nasopharyngeal Carcinoma: TTC40
    Article Snippet: .. Briefly, 1 μ g of DNA in 40 μ L was digested with either 10 units of methylation-sensitive restriction enzyme Hpa II (Fermentas) or 10 units of methylation-insensitive isoschizomer Msp I (Fermentas) at 37°C for 16 h. Aliquot of restriction-digested DNA (4 μ L) was amplified in a final volume of 50 μ L containing 1x PCR buffer, 50 pmol of a single primer MLG2 (5′-AACCCTCACCCTAACCCCGG-3′), 200 μ M of each dNTP, 2 mM of MgCl2 , and 1.25 units of Taq DNA polymerase (Fermentas). .. The reactions were carried out in a thermal cycler (Applied Biosystems 2720) for five cycles under low stringency (94°C for 30 s, 40°C for 60 s, and 72°C for 90 s) followed by 30 cycles under high stringency (95°C for 15 s, 55°C for 15 s, and 72°C for 60 s).

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 93
    Thermo Fisher electroporation solution
    Effect of cell aggregation on <t>electroporation</t> efficiency. ( a ) Images of fractionated T87-xGxGUS cells. T87-xGxGUS cell aggregates were fractionated into small (
    Electroporation Solution, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/electroporation solution/product/Thermo Fisher
    Average 93 stars, based on 12 article reviews
    Price from $9.99 to $1999.99
    electroporation solution - by Bioz Stars, 2020-07
    93/100 stars
      Buy from Supplier

    92
    Thermo Fisher mouse anti orf59
    ORF68 localizes within replication compartments and in cytoplasmic puncta of KSHV-infected cells. Reactivated and unreactivated BAC16.iSLK and TREx-BCBL1 cells were stained with antibodies to ORF68 (red), the viral replication compartment marker <t>ORF59</t> (green), and DAPI (blue). The rightmost column shows the merged images.
    Mouse Anti Orf59, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse anti orf59/product/Thermo Fisher
    Average 92 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mouse anti orf59 - by Bioz Stars, 2020-07
    92/100 stars
      Buy from Supplier

    91
    Thermo Fisher rnase r buffer
    Validation of HuR target circRNAs. (A) RT-qPCR analysis of HeLa circRNAs enriched in HuR IP samples compared with IgG samples; RNA levels were normalized to the background levels of GAPDH mRNA in each IP sample. (B) Example of PCR product validation; hsa_circ_0031288(CircPABPN1 ) was visualized on ethidium bromide-stained agarose gels to confirm the specificity of the circRNA amplification band (RT, reverse transcription). (C) Validation of circRNA PCR product from (B) by DNA sequencing analysis. All of the circRNAs in (A) were verified on agarose gels and many were further verified by sequencing. (D) RT-qPCR analysis to measure GAPDH mRNA, PABPN1 mRNA, and hsa_circ_0031288 ( CircPABPN1 ) with or without <t>RNase</t> R treatment; RNA levels in RNase R-treated samples were compared with mock (untreated) samples (using identical input RNA amounts). (E) Western blot analysis of HuR levels in the samples pulled down using control biotinylated GAPDH (3′UTR) and linear biotinylated CircPABPN1 . ‘Input’, HuR levels in the samples used for pulldown analysis. (F) Western blot analysis of HuR levels in pulldown assays using a biotinylated antisense oligomer targeting the junction of CircPABPN1 or a control biotinylated oligomer. Data in (A,D) are the means and ± SEM from three independent experiments. Data in (B,E,F) are representative of three independent experiments.
    Rnase R Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rnase r buffer/product/Thermo Fisher
    Average 91 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    rnase r buffer - by Bioz Stars, 2020-07
    91/100 stars
      Buy from Supplier

    88
    Thermo Fisher anti gpc1
    Tumor stage-specific analysis a, Table associated with receiver Operating Characteristic (ROC) curve analysis depicted in . b–f , ROC curve analysis for percent <t>GPC1</t> + crExos (red line), CA 19-9 serum levels (blue scattered line), exosomes concentration (black line) and exosomes size (scattered black line) in patients with carcinoma in situ (CIS) or stage I pancreatic cancer (n=5) (a), stage IIa pancreatic cancer (n=18) (b), stage IIb pancreatic cancer (n=117) (c), stage III pancreatic cancer (n=11) (d), and stage IV pancreas cancer (n=41) (e), compared to control (healthy donors (n=100) and patients with a benign pancreatic disease (n=26), total n=126). g . AUC: Area under the curve, CI: confidence interval. Figure 1f
    Anti Gpc1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti gpc1/product/Thermo Fisher
    Average 88 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    anti gpc1 - by Bioz Stars, 2020-07
    88/100 stars
      Buy from Supplier

    Image Search Results


    Effect of cell aggregation on electroporation efficiency. ( a ) Images of fractionated T87-xGxGUS cells. T87-xGxGUS cell aggregates were fractionated into small (

    Journal: Scientific Reports

    Article Title: A method using electroporation for the protein delivery of Cre recombinase into cultured Arabidopsis cells with an intact cell wall

    doi: 10.1038/s41598-018-38119-9

    Figure Lengend Snippet: Effect of cell aggregation on electroporation efficiency. ( a ) Images of fractionated T87-xGxGUS cells. T87-xGxGUS cell aggregates were fractionated into small (

    Article Snippet: On the day of electroporation, cells were collected in a tube and washed once with the electroporation solution: Opti-MEM I (Thermo Fisher Scientific), PBS (phosphate buffered saline, Thermo Fisher Scientific), NT1, B5 medium, deproteinized Opti-MEM I, MEM (Minimal Essential Medium, Thermo Fisher Scientific).

    Techniques: Electroporation

    Electroporation-mediated Cre protein delivery into T87 cells. ( a ) GUS staining of T87-xGxGUS cells electroporated using the indicated buffer. Cells were electroporated in the indicated buffer containing 1 μM Cre protein and 5 poring pulses of 375 V/cm for 10 ms. GUS staining was performed at 2 days after electroporation. Control indicates untreated T87-xGxGUS cells. Scale bar represents 200 μm. ( b ) Cytotoxicity of electroporation. Evans blue staining was performed at 1 hour after electroporation. Evans blue was extracted using 50% methanol/1% SDS and the absorbance at 595 nm was measured. Values shown are the mean ± SE of n = 3.

    Journal: Scientific Reports

    Article Title: A method using electroporation for the protein delivery of Cre recombinase into cultured Arabidopsis cells with an intact cell wall

    doi: 10.1038/s41598-018-38119-9

    Figure Lengend Snippet: Electroporation-mediated Cre protein delivery into T87 cells. ( a ) GUS staining of T87-xGxGUS cells electroporated using the indicated buffer. Cells were electroporated in the indicated buffer containing 1 μM Cre protein and 5 poring pulses of 375 V/cm for 10 ms. GUS staining was performed at 2 days after electroporation. Control indicates untreated T87-xGxGUS cells. Scale bar represents 200 μm. ( b ) Cytotoxicity of electroporation. Evans blue staining was performed at 1 hour after electroporation. Evans blue was extracted using 50% methanol/1% SDS and the absorbance at 595 nm was measured. Values shown are the mean ± SE of n = 3.

    Article Snippet: On the day of electroporation, cells were collected in a tube and washed once with the electroporation solution: Opti-MEM I (Thermo Fisher Scientific), PBS (phosphate buffered saline, Thermo Fisher Scientific), NT1, B5 medium, deproteinized Opti-MEM I, MEM (Minimal Essential Medium, Thermo Fisher Scientific).

    Techniques: Electroporation, Staining, Mass Spectrometry

    Effect of poring pulse conditions on electroporation efficiency. ( a – c ) Normalized GUS activity as determined by the fluorescent measurement of catalyzed CMUG in electroporated T87-xGxGUS cells using different poring pulse conditions. ( a ) Field strength (250, 375, 500, or 625 V/cm), ( b ) duration (2, 5, 10, or 25 ms), and ( c ) number (1, 2, 5, or 9) of poring pulses were changed from the standard poring pulse conditions (375 V/cm, 10 ms, 5 times). In all cases, 1 μM Cre protein dissolved in Opti-MEMI was used for electroporation. Control indicates untreated T87-xGxGUS cells. Two days after electroporation, GUS activity was measured and normalized to the amount of chlorophyll. Values shown are the mean ± SE of n = 3.

    Journal: Scientific Reports

    Article Title: A method using electroporation for the protein delivery of Cre recombinase into cultured Arabidopsis cells with an intact cell wall

    doi: 10.1038/s41598-018-38119-9

    Figure Lengend Snippet: Effect of poring pulse conditions on electroporation efficiency. ( a – c ) Normalized GUS activity as determined by the fluorescent measurement of catalyzed CMUG in electroporated T87-xGxGUS cells using different poring pulse conditions. ( a ) Field strength (250, 375, 500, or 625 V/cm), ( b ) duration (2, 5, 10, or 25 ms), and ( c ) number (1, 2, 5, or 9) of poring pulses were changed from the standard poring pulse conditions (375 V/cm, 10 ms, 5 times). In all cases, 1 μM Cre protein dissolved in Opti-MEMI was used for electroporation. Control indicates untreated T87-xGxGUS cells. Two days after electroporation, GUS activity was measured and normalized to the amount of chlorophyll. Values shown are the mean ± SE of n = 3.

    Article Snippet: On the day of electroporation, cells were collected in a tube and washed once with the electroporation solution: Opti-MEM I (Thermo Fisher Scientific), PBS (phosphate buffered saline, Thermo Fisher Scientific), NT1, B5 medium, deproteinized Opti-MEM I, MEM (Minimal Essential Medium, Thermo Fisher Scientific).

    Techniques: Electroporation, Activity Assay, Mass Spectrometry

    Effect of Cre protein concentration on electroporation efficiency. ( a ) GUS staining of T87-xGxGUS cells electroporated with different concentrations of Cre protein. Cells were electroporated with the indicated concentration of Cre protein in Opti-MEMI using 5 poring pulses of 375 V/cm for 10 ms. GUS staining was performed 2 days after electroporation. Control indicates untreated T87-xGxGUS cells. Scale bar represents 200 μm. ( b ) Normalized GUS activity as determined by the level of fluorescence of catalyzed CMUG in electroporated cells with different concentrations of Cre protein. Cells were electroporated with 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0, or 5.0 μM of Cre protein in Opti-MEMI using 5 poring pulses of 375 V/cm for 10 ms. Two days after electroporation, GUS activity was measured and normalized to the chlorophyll amount. Values shown are the mean ± SE of n = 3. ( c ) Cytotoxicity of electroporation. Evans blue staining was performed at 1 hour after electroporation. Evans blue was extracted using 50% methanol/1% SDS and the absorbance at 595 nm was measured. Control indicates untreated T87-xGxGUS cells. Values shown are the mean ± SE of n = 3.

    Journal: Scientific Reports

    Article Title: A method using electroporation for the protein delivery of Cre recombinase into cultured Arabidopsis cells with an intact cell wall

    doi: 10.1038/s41598-018-38119-9

    Figure Lengend Snippet: Effect of Cre protein concentration on electroporation efficiency. ( a ) GUS staining of T87-xGxGUS cells electroporated with different concentrations of Cre protein. Cells were electroporated with the indicated concentration of Cre protein in Opti-MEMI using 5 poring pulses of 375 V/cm for 10 ms. GUS staining was performed 2 days after electroporation. Control indicates untreated T87-xGxGUS cells. Scale bar represents 200 μm. ( b ) Normalized GUS activity as determined by the level of fluorescence of catalyzed CMUG in electroporated cells with different concentrations of Cre protein. Cells were electroporated with 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0, or 5.0 μM of Cre protein in Opti-MEMI using 5 poring pulses of 375 V/cm for 10 ms. Two days after electroporation, GUS activity was measured and normalized to the chlorophyll amount. Values shown are the mean ± SE of n = 3. ( c ) Cytotoxicity of electroporation. Evans blue staining was performed at 1 hour after electroporation. Evans blue was extracted using 50% methanol/1% SDS and the absorbance at 595 nm was measured. Control indicates untreated T87-xGxGUS cells. Values shown are the mean ± SE of n = 3.

    Article Snippet: On the day of electroporation, cells were collected in a tube and washed once with the electroporation solution: Opti-MEM I (Thermo Fisher Scientific), PBS (phosphate buffered saline, Thermo Fisher Scientific), NT1, B5 medium, deproteinized Opti-MEM I, MEM (Minimal Essential Medium, Thermo Fisher Scientific).

    Techniques: Protein Concentration, Electroporation, Staining, Concentration Assay, Mass Spectrometry, Activity Assay, Fluorescence

    Quantification of protein delivery efficiency. ( a ) GUS staining of protoplasts constructed from electroporated T87-xGxGUS cells. Cells were electroporated with 5 µM of Cre protein, dissolved in Opti-MEMI, using 5 poring pulses of 375 V/cm for 10 ms. Protoplasts were constructed and stained 2 days after electroporation. Control indicates unelectroporated T87-xGxGUS cells. Scale bar represents 40 μm. ( b ) Quantification of the percentage of GUS positive cells from ( a ). Values shown are the mean ± SE of n = 3. In total, 254 and 288 cells were used for the quantification of control and electroporation samples, respectively. ( c ) Schematic of Cre reporter design before and after Cre-mediated recombination. Black arrows indicate primer binding sites. GFP and GUS indicate green fluorescent protein and ß-glucuronidase, respectively. (d) Agarose gel electrophoresis of genomic DNA PCR products from T87-xGxGUS cells. The 1307 bp and 151 bp fragments represent the reporter gene cassette before and after Cre-mediated recombination, respectively. A full-length gel image is presented in Supplementary Figure 6 .

    Journal: Scientific Reports

    Article Title: A method using electroporation for the protein delivery of Cre recombinase into cultured Arabidopsis cells with an intact cell wall

    doi: 10.1038/s41598-018-38119-9

    Figure Lengend Snippet: Quantification of protein delivery efficiency. ( a ) GUS staining of protoplasts constructed from electroporated T87-xGxGUS cells. Cells were electroporated with 5 µM of Cre protein, dissolved in Opti-MEMI, using 5 poring pulses of 375 V/cm for 10 ms. Protoplasts were constructed and stained 2 days after electroporation. Control indicates unelectroporated T87-xGxGUS cells. Scale bar represents 40 μm. ( b ) Quantification of the percentage of GUS positive cells from ( a ). Values shown are the mean ± SE of n = 3. In total, 254 and 288 cells were used for the quantification of control and electroporation samples, respectively. ( c ) Schematic of Cre reporter design before and after Cre-mediated recombination. Black arrows indicate primer binding sites. GFP and GUS indicate green fluorescent protein and ß-glucuronidase, respectively. (d) Agarose gel electrophoresis of genomic DNA PCR products from T87-xGxGUS cells. The 1307 bp and 151 bp fragments represent the reporter gene cassette before and after Cre-mediated recombination, respectively. A full-length gel image is presented in Supplementary Figure 6 .

    Article Snippet: On the day of electroporation, cells were collected in a tube and washed once with the electroporation solution: Opti-MEM I (Thermo Fisher Scientific), PBS (phosphate buffered saline, Thermo Fisher Scientific), NT1, B5 medium, deproteinized Opti-MEM I, MEM (Minimal Essential Medium, Thermo Fisher Scientific).

    Techniques: Staining, Construct, Mass Spectrometry, Electroporation, Binding Assay, Agarose Gel Electrophoresis, Polymerase Chain Reaction

    ORF68 localizes within replication compartments and in cytoplasmic puncta of KSHV-infected cells. Reactivated and unreactivated BAC16.iSLK and TREx-BCBL1 cells were stained with antibodies to ORF68 (red), the viral replication compartment marker ORF59 (green), and DAPI (blue). The rightmost column shows the merged images.

    Journal: Journal of Virology

    Article Title: Kaposi's Sarcoma-Associated Herpesvirus ORF68 Is a DNA Binding Protein Required for Viral Genome Cleavage and Packaging

    doi: 10.1128/JVI.00840-18

    Figure Lengend Snippet: ORF68 localizes within replication compartments and in cytoplasmic puncta of KSHV-infected cells. Reactivated and unreactivated BAC16.iSLK and TREx-BCBL1 cells were stained with antibodies to ORF68 (red), the viral replication compartment marker ORF59 (green), and DAPI (blue). The rightmost column shows the merged images.

    Article Snippet: The cells were then incubated with rabbit anti-ORF68 and mouse anti-ORF59 (Advanced Biotechnologies) antibodies (diluted 1:100 in blocking buffer) overnight at 4°C, washed with PBS, incubated with secondary antibody (AlexaFluor594 or DyLight650, 1:1,000 in BSA blocking buffer) for 1 h at 37°C, and mounted using VectaShield HardSet with DAPI (4′,6′-diamidino-2-phenylindole; Thermo Fisher).

    Techniques: Infection, Staining, Marker

    Characterization of the ORF68-deficient (ORF68 PTC ) mutant virus. (A) Diagram depicting the genetic locus of ORF68 and the mutations inserted into the ORF68 PTC virus. (B) Western blots showing expression of the early ORF59 protein and the late K8.1 protein in WT, ORF68 PTC , and ORF68 PTC -MR viruses. Histone H3 served as a loading control. (C) DNA replication was measured by qPCR of the viral genome before and after induction of the lytic cycle. (D) Progeny virion production was assayed by supernatant transfer and flow cytometry of target cells.

    Journal: Journal of Virology

    Article Title: Kaposi's Sarcoma-Associated Herpesvirus ORF68 Is a DNA Binding Protein Required for Viral Genome Cleavage and Packaging

    doi: 10.1128/JVI.00840-18

    Figure Lengend Snippet: Characterization of the ORF68-deficient (ORF68 PTC ) mutant virus. (A) Diagram depicting the genetic locus of ORF68 and the mutations inserted into the ORF68 PTC virus. (B) Western blots showing expression of the early ORF59 protein and the late K8.1 protein in WT, ORF68 PTC , and ORF68 PTC -MR viruses. Histone H3 served as a loading control. (C) DNA replication was measured by qPCR of the viral genome before and after induction of the lytic cycle. (D) Progeny virion production was assayed by supernatant transfer and flow cytometry of target cells.

    Article Snippet: The cells were then incubated with rabbit anti-ORF68 and mouse anti-ORF59 (Advanced Biotechnologies) antibodies (diluted 1:100 in blocking buffer) overnight at 4°C, washed with PBS, incubated with secondary antibody (AlexaFluor594 or DyLight650, 1:1,000 in BSA blocking buffer) for 1 h at 37°C, and mounted using VectaShield HardSet with DAPI (4′,6′-diamidino-2-phenylindole; Thermo Fisher).

    Techniques: Mutagenesis, Western Blot, Expressing, Real-time Polymerase Chain Reaction, Flow Cytometry, Cytometry

    Characterization of the expression of KSHV ORF68. (A) Western blot showing the expression kinetics of ORF68 relative to the early protein ORF59 and the late protein K8.1 in iSLK.BAC16 cells after reactivation with doxycycline (Dox). GAPDH served as a loading control. (B) Anti-ORF68 Western blot of lysates of HEK293T cells transfected with plasmids expressing either the extended (ORF68-Extended) or shorter form of ORF68, next to lysates of lytically reactivated iSLK.BAC16 and TREx-BCBL1 cells. Different amounts of lysate were loaded into each lane to account for differing ORF68 expression levels. (C) Diagram showing the genomic locus of ORF68, showing the two annotated forms, which differ by a 78-aa N-terminal extension.

    Journal: Journal of Virology

    Article Title: Kaposi's Sarcoma-Associated Herpesvirus ORF68 Is a DNA Binding Protein Required for Viral Genome Cleavage and Packaging

    doi: 10.1128/JVI.00840-18

    Figure Lengend Snippet: Characterization of the expression of KSHV ORF68. (A) Western blot showing the expression kinetics of ORF68 relative to the early protein ORF59 and the late protein K8.1 in iSLK.BAC16 cells after reactivation with doxycycline (Dox). GAPDH served as a loading control. (B) Anti-ORF68 Western blot of lysates of HEK293T cells transfected with plasmids expressing either the extended (ORF68-Extended) or shorter form of ORF68, next to lysates of lytically reactivated iSLK.BAC16 and TREx-BCBL1 cells. Different amounts of lysate were loaded into each lane to account for differing ORF68 expression levels. (C) Diagram showing the genomic locus of ORF68, showing the two annotated forms, which differ by a 78-aa N-terminal extension.

    Article Snippet: The cells were then incubated with rabbit anti-ORF68 and mouse anti-ORF59 (Advanced Biotechnologies) antibodies (diluted 1:100 in blocking buffer) overnight at 4°C, washed with PBS, incubated with secondary antibody (AlexaFluor594 or DyLight650, 1:1,000 in BSA blocking buffer) for 1 h at 37°C, and mounted using VectaShield HardSet with DAPI (4′,6′-diamidino-2-phenylindole; Thermo Fisher).

    Techniques: Expressing, Western Blot, Transfection

    Validation of HuR target circRNAs. (A) RT-qPCR analysis of HeLa circRNAs enriched in HuR IP samples compared with IgG samples; RNA levels were normalized to the background levels of GAPDH mRNA in each IP sample. (B) Example of PCR product validation; hsa_circ_0031288(CircPABPN1 ) was visualized on ethidium bromide-stained agarose gels to confirm the specificity of the circRNA amplification band (RT, reverse transcription). (C) Validation of circRNA PCR product from (B) by DNA sequencing analysis. All of the circRNAs in (A) were verified on agarose gels and many were further verified by sequencing. (D) RT-qPCR analysis to measure GAPDH mRNA, PABPN1 mRNA, and hsa_circ_0031288 ( CircPABPN1 ) with or without RNase R treatment; RNA levels in RNase R-treated samples were compared with mock (untreated) samples (using identical input RNA amounts). (E) Western blot analysis of HuR levels in the samples pulled down using control biotinylated GAPDH (3′UTR) and linear biotinylated CircPABPN1 . ‘Input’, HuR levels in the samples used for pulldown analysis. (F) Western blot analysis of HuR levels in pulldown assays using a biotinylated antisense oligomer targeting the junction of CircPABPN1 or a control biotinylated oligomer. Data in (A,D) are the means and ± SEM from three independent experiments. Data in (B,E,F) are representative of three independent experiments.

    Journal: RNA Biology

    Article Title: Identification of HuR target circular RNAs uncovers suppression of PABPN1 translation by CircPABPN1

    doi: 10.1080/15476286.2017.1279788

    Figure Lengend Snippet: Validation of HuR target circRNAs. (A) RT-qPCR analysis of HeLa circRNAs enriched in HuR IP samples compared with IgG samples; RNA levels were normalized to the background levels of GAPDH mRNA in each IP sample. (B) Example of PCR product validation; hsa_circ_0031288(CircPABPN1 ) was visualized on ethidium bromide-stained agarose gels to confirm the specificity of the circRNA amplification band (RT, reverse transcription). (C) Validation of circRNA PCR product from (B) by DNA sequencing analysis. All of the circRNAs in (A) were verified on agarose gels and many were further verified by sequencing. (D) RT-qPCR analysis to measure GAPDH mRNA, PABPN1 mRNA, and hsa_circ_0031288 ( CircPABPN1 ) with or without RNase R treatment; RNA levels in RNase R-treated samples were compared with mock (untreated) samples (using identical input RNA amounts). (E) Western blot analysis of HuR levels in the samples pulled down using control biotinylated GAPDH (3′UTR) and linear biotinylated CircPABPN1 . ‘Input’, HuR levels in the samples used for pulldown analysis. (F) Western blot analysis of HuR levels in pulldown assays using a biotinylated antisense oligomer targeting the junction of CircPABPN1 or a control biotinylated oligomer. Data in (A,D) are the means and ± SEM from three independent experiments. Data in (B,E,F) are representative of three independent experiments.

    Article Snippet: One µg total RNA was either left untreated (mock) or treated with 10 units of RNase R (RNR07250, Epicentre) in the presence of 1× RNase R buffer, 20 units of RiboLock RNase Inhibitor (Thermo Scientific), and incubated for 30 min at 37°C.

    Techniques: Quantitative RT-PCR, Polymerase Chain Reaction, Staining, Amplification, DNA Sequencing, Sequencing, Western Blot

    Transcriptome-wide identification of HuR-associated circRNAs. (A,B) Schematic of the strategy used to identify globally HuR-interacting circRNAs in HeLa cells (A). Following cell lysis, RIP analysis was carried out using anti-HuR or control IgG antibodies; the presence of HuR in the IP samples was assessed by Western blot analysis [(B); HC, heavy IgG chain]. Total RNA was isolated and digested with RNase R, and circRNAs present in the sample were identified using circRNA microarrays (Arraystar). (C) Partial list of circRNAs highly enriched in HuR IP relative to IgG IP as identified in microarrays (n = 3).

    Journal: RNA Biology

    Article Title: Identification of HuR target circular RNAs uncovers suppression of PABPN1 translation by CircPABPN1

    doi: 10.1080/15476286.2017.1279788

    Figure Lengend Snippet: Transcriptome-wide identification of HuR-associated circRNAs. (A,B) Schematic of the strategy used to identify globally HuR-interacting circRNAs in HeLa cells (A). Following cell lysis, RIP analysis was carried out using anti-HuR or control IgG antibodies; the presence of HuR in the IP samples was assessed by Western blot analysis [(B); HC, heavy IgG chain]. Total RNA was isolated and digested with RNase R, and circRNAs present in the sample were identified using circRNA microarrays (Arraystar). (C) Partial list of circRNAs highly enriched in HuR IP relative to IgG IP as identified in microarrays (n = 3).

    Article Snippet: One µg total RNA was either left untreated (mock) or treated with 10 units of RNase R (RNR07250, Epicentre) in the presence of 1× RNase R buffer, 20 units of RiboLock RNase Inhibitor (Thermo Scientific), and incubated for 30 min at 37°C.

    Techniques: Lysis, Western Blot, Isolation

    Tumor stage-specific analysis a, Table associated with receiver Operating Characteristic (ROC) curve analysis depicted in . b–f , ROC curve analysis for percent GPC1 + crExos (red line), CA 19-9 serum levels (blue scattered line), exosomes concentration (black line) and exosomes size (scattered black line) in patients with carcinoma in situ (CIS) or stage I pancreatic cancer (n=5) (a), stage IIa pancreatic cancer (n=18) (b), stage IIb pancreatic cancer (n=117) (c), stage III pancreatic cancer (n=11) (d), and stage IV pancreas cancer (n=41) (e), compared to control (healthy donors (n=100) and patients with a benign pancreatic disease (n=26), total n=126). g . AUC: Area under the curve, CI: confidence interval. Figure 1f

    Journal: Nature

    Article Title: Glypican1 identifies cancer exosomes and facilitates early detection of cancer

    doi: 10.1038/nature14581

    Figure Lengend Snippet: Tumor stage-specific analysis a, Table associated with receiver Operating Characteristic (ROC) curve analysis depicted in . b–f , ROC curve analysis for percent GPC1 + crExos (red line), CA 19-9 serum levels (blue scattered line), exosomes concentration (black line) and exosomes size (scattered black line) in patients with carcinoma in situ (CIS) or stage I pancreatic cancer (n=5) (a), stage IIa pancreatic cancer (n=18) (b), stage IIb pancreatic cancer (n=117) (c), stage III pancreatic cancer (n=11) (d), and stage IV pancreas cancer (n=41) (e), compared to control (healthy donors (n=100) and patients with a benign pancreatic disease (n=26), total n=126). g . AUC: Area under the curve, CI: confidence interval. Figure 1f

    Article Snippet: Exosomes-bound beads were washed 1 time in 1X PBS/2% BSA and centrifuge for 1 min at 10,000 rpm, blocked with 10% BSA with rotation at room temperature for 30 min, washed a second time in 1X PBS/2% BSA and centrifuged for 1 min at 10,000 rpm, and incubated with anti-GPC1 (PIPA528055, Thermo-Scientific, 3 μl of antibody in 20 μl of 2%BSA/1X PBS) during 30 min rotating at 4°C.

    Techniques: Concentration Assay, In Situ

    GPC1 + crExos is a non-invasive biomarker for pancreas cancer a , Percent beads with GPC1 + crExos in healthy donors (n=100), breast cancer patients (n=32) and patients with PDAC (n=190; ANOVA, post-hoc Tamhane T 2 , **** P

    Journal: Nature

    Article Title: Glypican1 identifies cancer exosomes and facilitates early detection of cancer

    doi: 10.1038/nature14581

    Figure Lengend Snippet: GPC1 + crExos is a non-invasive biomarker for pancreas cancer a , Percent beads with GPC1 + crExos in healthy donors (n=100), breast cancer patients (n=32) and patients with PDAC (n=190; ANOVA, post-hoc Tamhane T 2 , **** P

    Article Snippet: Exosomes-bound beads were washed 1 time in 1X PBS/2% BSA and centrifuge for 1 min at 10,000 rpm, blocked with 10% BSA with rotation at room temperature for 30 min, washed a second time in 1X PBS/2% BSA and centrifuged for 1 min at 10,000 rpm, and incubated with anti-GPC1 (PIPA528055, Thermo-Scientific, 3 μl of antibody in 20 μl of 2%BSA/1X PBS) during 30 min rotating at 4°C.

    Techniques: Biomarker Assay

    Exosomes isolation a , Exosomes concentration and size distribution by NanoSight® analysis of culture supernatant from NIH/3T3, MCF10A, HDF, MDA-MB-231 and E10 cells. Size mode: 105 nanometers (nm; 3 technical replicates). b , Transmission electron microscopy (TEM) micrograph of MDA-MB-231-derived exosomes. Upper right image shows a digitally zoomed inset. c , TEM micrograph of MDA-MB-231-derived exosomes following immunogold labeling for CD9. Gold particles are depicted as black dots. Upper right image shows a digitally zoomed inset. d , Immunoblot of flotillin1 and CD81 in exosomal proteins extracted from culture supernatant of E10, NIH/3T3, MDA-MB-231, MCF10A and HDF cells. e , RT–qPCR measurement of GPC1 mRNA levels in HMEL, HDF, HMLE, MCF7, MDA-MB-231, T3M4, Panc-1, MIA Paca2. Results are shown as mean ± standard deviation; n=3, 3 biological replicates, with 3 technical replicates each. f , Immunoblot of GPC1 in HMEL, HDF, HMLE, MCF7, MDA-MB-231, T3M4, Panc-1 and MIA Paca2 cell lysates (upper panel). β-actin was used as a loading control (lower panel). g , Immunoblot of GPC1 in exosomal protein lysates derived from the culture supernatant of 3 non-tumorigenic cell lines (HDF, HMEL, HMLE) and 5 tumorigenic cell lines (MCF7, MDA-MB-231, T3M4, Panc-1, MIA Paca2) (upper panel). Immunoblot of flotillin1 was used as loading control (lower panel). h , Immunoblot of flotillin1 in exosomal protein lysates from the culture supernatant of MDA-MB-231 and T3M4 following sucrose gradient purification. The protein content is assayed in each of the density layers listed.

    Journal: Nature

    Article Title: Glypican1 identifies cancer exosomes and facilitates early detection of cancer

    doi: 10.1038/nature14581

    Figure Lengend Snippet: Exosomes isolation a , Exosomes concentration and size distribution by NanoSight® analysis of culture supernatant from NIH/3T3, MCF10A, HDF, MDA-MB-231 and E10 cells. Size mode: 105 nanometers (nm; 3 technical replicates). b , Transmission electron microscopy (TEM) micrograph of MDA-MB-231-derived exosomes. Upper right image shows a digitally zoomed inset. c , TEM micrograph of MDA-MB-231-derived exosomes following immunogold labeling for CD9. Gold particles are depicted as black dots. Upper right image shows a digitally zoomed inset. d , Immunoblot of flotillin1 and CD81 in exosomal proteins extracted from culture supernatant of E10, NIH/3T3, MDA-MB-231, MCF10A and HDF cells. e , RT–qPCR measurement of GPC1 mRNA levels in HMEL, HDF, HMLE, MCF7, MDA-MB-231, T3M4, Panc-1, MIA Paca2. Results are shown as mean ± standard deviation; n=3, 3 biological replicates, with 3 technical replicates each. f , Immunoblot of GPC1 in HMEL, HDF, HMLE, MCF7, MDA-MB-231, T3M4, Panc-1 and MIA Paca2 cell lysates (upper panel). β-actin was used as a loading control (lower panel). g , Immunoblot of GPC1 in exosomal protein lysates derived from the culture supernatant of 3 non-tumorigenic cell lines (HDF, HMEL, HMLE) and 5 tumorigenic cell lines (MCF7, MDA-MB-231, T3M4, Panc-1, MIA Paca2) (upper panel). Immunoblot of flotillin1 was used as loading control (lower panel). h , Immunoblot of flotillin1 in exosomal protein lysates from the culture supernatant of MDA-MB-231 and T3M4 following sucrose gradient purification. The protein content is assayed in each of the density layers listed.

    Article Snippet: Exosomes-bound beads were washed 1 time in 1X PBS/2% BSA and centrifuge for 1 min at 10,000 rpm, blocked with 10% BSA with rotation at room temperature for 30 min, washed a second time in 1X PBS/2% BSA and centrifuged for 1 min at 10,000 rpm, and incubated with anti-GPC1 (PIPA528055, Thermo-Scientific, 3 μl of antibody in 20 μl of 2%BSA/1X PBS) during 30 min rotating at 4°C.

    Techniques: Isolation, Concentration Assay, Multiple Displacement Amplification, Transmission Assay, Electron Microscopy, Transmission Electron Microscopy, Derivative Assay, Labeling, Quantitative RT-PCR, Standard Deviation, Purification

    GPC1 + crExosomes are derived from cancer cells in tumor-bearing mice a , Longitudinal blood collection: nude mice with orthotopic MDA-MB-231 tumors (n=4 mice). b , Percentage of beads with GPC1 + exosomes harvested from systemic circulation (crExos) plotted against average tumor volume (n=4 mice, each sample analyzed in technical triplicates for GPC1). ANOVA, post-hoc Tamhane T 2 , ** P

    Journal: Nature

    Article Title: Glypican1 identifies cancer exosomes and facilitates early detection of cancer

    doi: 10.1038/nature14581

    Figure Lengend Snippet: GPC1 + crExosomes are derived from cancer cells in tumor-bearing mice a , Longitudinal blood collection: nude mice with orthotopic MDA-MB-231 tumors (n=4 mice). b , Percentage of beads with GPC1 + exosomes harvested from systemic circulation (crExos) plotted against average tumor volume (n=4 mice, each sample analyzed in technical triplicates for GPC1). ANOVA, post-hoc Tamhane T 2 , ** P

    Article Snippet: Exosomes-bound beads were washed 1 time in 1X PBS/2% BSA and centrifuge for 1 min at 10,000 rpm, blocked with 10% BSA with rotation at room temperature for 30 min, washed a second time in 1X PBS/2% BSA and centrifuged for 1 min at 10,000 rpm, and incubated with anti-GPC1 (PIPA528055, Thermo-Scientific, 3 μl of antibody in 20 μl of 2%BSA/1X PBS) during 30 min rotating at 4°C.

    Techniques: Derivative Assay, Mouse Assay, Multiple Displacement Amplification

    PDAC GEMM cross sectional studies a , Representative micrographs of H E stained pancreas from 16 days old control mice (left panel) and PKT mice presenting with (right panel, encircled) and without (middle panel) PanIN lesions. Scale bars: 100 μm. b , Ct values following qPCR analyses for oncogenic KRAS G12D , wild-type KRAS and 18S internal control RNA from exosomes of 44–48 days old PKT mice serum segregated using FACS for GPC1 + bead bound exos (red) and GPC1 − bead bound exos (blue). Data is presented as the mean ± standard deviation.

    Journal: Nature

    Article Title: Glypican1 identifies cancer exosomes and facilitates early detection of cancer

    doi: 10.1038/nature14581

    Figure Lengend Snippet: PDAC GEMM cross sectional studies a , Representative micrographs of H E stained pancreas from 16 days old control mice (left panel) and PKT mice presenting with (right panel, encircled) and without (middle panel) PanIN lesions. Scale bars: 100 μm. b , Ct values following qPCR analyses for oncogenic KRAS G12D , wild-type KRAS and 18S internal control RNA from exosomes of 44–48 days old PKT mice serum segregated using FACS for GPC1 + bead bound exos (red) and GPC1 − bead bound exos (blue). Data is presented as the mean ± standard deviation.

    Article Snippet: Exosomes-bound beads were washed 1 time in 1X PBS/2% BSA and centrifuge for 1 min at 10,000 rpm, blocked with 10% BSA with rotation at room temperature for 30 min, washed a second time in 1X PBS/2% BSA and centrifuged for 1 min at 10,000 rpm, and incubated with anti-GPC1 (PIPA528055, Thermo-Scientific, 3 μl of antibody in 20 μl of 2%BSA/1X PBS) during 30 min rotating at 4°C.

    Techniques: Staining, Mouse Assay, Real-time Polymerase Chain Reaction, FACS, Standard Deviation

    Raw scatter dot plot depicting flow cytometry analyses of beads with GPC1 + bound exosomes a , Scatter plots and histogram of flow cytometry analyses of serum exosomes on beads of a representative healthy control (left panels are secondary antibody only; right panels are GPC1 antibody and secondary antibody). b , Scatter plots and histogram of flow cytometry analysis of serum exosomes on beads of a representative pancreas cancer sample (left panels are secondary antibody only; right panels are with GPC1 antibody and secondary antibody).

    Journal: Nature

    Article Title: Glypican1 identifies cancer exosomes and facilitates early detection of cancer

    doi: 10.1038/nature14581

    Figure Lengend Snippet: Raw scatter dot plot depicting flow cytometry analyses of beads with GPC1 + bound exosomes a , Scatter plots and histogram of flow cytometry analyses of serum exosomes on beads of a representative healthy control (left panels are secondary antibody only; right panels are GPC1 antibody and secondary antibody). b , Scatter plots and histogram of flow cytometry analysis of serum exosomes on beads of a representative pancreas cancer sample (left panels are secondary antibody only; right panels are with GPC1 antibody and secondary antibody).

    Article Snippet: Exosomes-bound beads were washed 1 time in 1X PBS/2% BSA and centrifuge for 1 min at 10,000 rpm, blocked with 10% BSA with rotation at room temperature for 30 min, washed a second time in 1X PBS/2% BSA and centrifuged for 1 min at 10,000 rpm, and incubated with anti-GPC1 (PIPA528055, Thermo-Scientific, 3 μl of antibody in 20 μl of 2%BSA/1X PBS) during 30 min rotating at 4°C.

    Techniques: Flow Cytometry, Cytometry

    GPC1 + circulating exosomes predict pancreas cancer in GEMM a , Longitudinal blood collection from control and PKT mice: 4 (n=6 and n=7, respectively), 5 (n=6 and n=7), 6 (n=6 and n=6), 7 (n=6 and n=6) and 8 (n=2 and n=3) weeks of age. b , Percent beads with GPC1 + crExos from PKT (red) and control (blue) mice; ANOVA, post-hoc Tukey-Kramer test, **** P

    Journal: Nature

    Article Title: Glypican1 identifies cancer exosomes and facilitates early detection of cancer

    doi: 10.1038/nature14581

    Figure Lengend Snippet: GPC1 + circulating exosomes predict pancreas cancer in GEMM a , Longitudinal blood collection from control and PKT mice: 4 (n=6 and n=7, respectively), 5 (n=6 and n=7), 6 (n=6 and n=6), 7 (n=6 and n=6) and 8 (n=2 and n=3) weeks of age. b , Percent beads with GPC1 + crExos from PKT (red) and control (blue) mice; ANOVA, post-hoc Tukey-Kramer test, **** P

    Article Snippet: Exosomes-bound beads were washed 1 time in 1X PBS/2% BSA and centrifuge for 1 min at 10,000 rpm, blocked with 10% BSA with rotation at room temperature for 30 min, washed a second time in 1X PBS/2% BSA and centrifuged for 1 min at 10,000 rpm, and incubated with anti-GPC1 (PIPA528055, Thermo-Scientific, 3 μl of antibody in 20 μl of 2%BSA/1X PBS) during 30 min rotating at 4°C.

    Techniques: Mouse Assay

    Levels of circulating GPC1 + exosomes inform pancreas cancer resection outcome a , Longitudinal blood collection pre- (pre-op) and post-operatively (day 7). b , Percent beads with GPC1 + crExos from patients with BPD (n=4), PCPL (n=4) or PDAC (n=29) (paired two-tailed Student’s t -test, ** P

    Journal: Nature

    Article Title: Glypican1 identifies cancer exosomes and facilitates early detection of cancer

    doi: 10.1038/nature14581

    Figure Lengend Snippet: Levels of circulating GPC1 + exosomes inform pancreas cancer resection outcome a , Longitudinal blood collection pre- (pre-op) and post-operatively (day 7). b , Percent beads with GPC1 + crExos from patients with BPD (n=4), PCPL (n=4) or PDAC (n=29) (paired two-tailed Student’s t -test, ** P

    Article Snippet: Exosomes-bound beads were washed 1 time in 1X PBS/2% BSA and centrifuge for 1 min at 10,000 rpm, blocked with 10% BSA with rotation at room temperature for 30 min, washed a second time in 1X PBS/2% BSA and centrifuged for 1 min at 10,000 rpm, and incubated with anti-GPC1 (PIPA528055, Thermo-Scientific, 3 μl of antibody in 20 μl of 2%BSA/1X PBS) during 30 min rotating at 4°C.

    Techniques: Two Tailed Test

    Longitudinal human study a , Scatter plots of percent beads with GPC1 + crExos by flow cytometry in patients with pancreas cancer. Patients are divided based on metastatic disease (non-metastatic lesions, lymph node metastases and distant metastases). ANOVA, post-hoc Tukey-Kramer test, * P

    Journal: Nature

    Article Title: Glypican1 identifies cancer exosomes and facilitates early detection of cancer

    doi: 10.1038/nature14581

    Figure Lengend Snippet: Longitudinal human study a , Scatter plots of percent beads with GPC1 + crExos by flow cytometry in patients with pancreas cancer. Patients are divided based on metastatic disease (non-metastatic lesions, lymph node metastases and distant metastases). ANOVA, post-hoc Tukey-Kramer test, * P

    Article Snippet: Exosomes-bound beads were washed 1 time in 1X PBS/2% BSA and centrifuge for 1 min at 10,000 rpm, blocked with 10% BSA with rotation at room temperature for 30 min, washed a second time in 1X PBS/2% BSA and centrifuged for 1 min at 10,000 rpm, and incubated with anti-GPC1 (PIPA528055, Thermo-Scientific, 3 μl of antibody in 20 μl of 2%BSA/1X PBS) during 30 min rotating at 4°C.

    Techniques: Flow Cytometry, Cytometry

    GPC1 is present specifically on cancer exosomes a , Venn diagram of exosomal proteins from NIH/3T3 (blue), MCF 10A (red), HDF (green), E10 (yellow) and MDA-MB-231 (purple) cells. 48 proteins were exclusively detected in exosomes from MDA-MB-231 cells (n=3 protein samples, technical replicates). b , Transmission electron micrographs (TEM; left image) and immunogold TEM (right image) of GPC1. Upper right: digitally zoomed inset (n=2 experiments). c , Diagram of flow cytometry experiment to detect GPC1 on the surface of exosomes bound to beads. d , TEM of bead-bound exosomes and immunogold labeling of GPC1 (n=2 biological replicates). e , Percent beads with GPC1 + exosomes from cancer cells (red) and non-tumorigenic cells (black). f , Flow cytometry analyses of percent beads with GPC1 + exosomes from indicated cell lines (n=2 biological replicates). Negative control: secondary antibody only.

    Journal: Nature

    Article Title: Glypican1 identifies cancer exosomes and facilitates early detection of cancer

    doi: 10.1038/nature14581

    Figure Lengend Snippet: GPC1 is present specifically on cancer exosomes a , Venn diagram of exosomal proteins from NIH/3T3 (blue), MCF 10A (red), HDF (green), E10 (yellow) and MDA-MB-231 (purple) cells. 48 proteins were exclusively detected in exosomes from MDA-MB-231 cells (n=3 protein samples, technical replicates). b , Transmission electron micrographs (TEM; left image) and immunogold TEM (right image) of GPC1. Upper right: digitally zoomed inset (n=2 experiments). c , Diagram of flow cytometry experiment to detect GPC1 on the surface of exosomes bound to beads. d , TEM of bead-bound exosomes and immunogold labeling of GPC1 (n=2 biological replicates). e , Percent beads with GPC1 + exosomes from cancer cells (red) and non-tumorigenic cells (black). f , Flow cytometry analyses of percent beads with GPC1 + exosomes from indicated cell lines (n=2 biological replicates). Negative control: secondary antibody only.

    Article Snippet: Exosomes-bound beads were washed 1 time in 1X PBS/2% BSA and centrifuge for 1 min at 10,000 rpm, blocked with 10% BSA with rotation at room temperature for 30 min, washed a second time in 1X PBS/2% BSA and centrifuged for 1 min at 10,000 rpm, and incubated with anti-GPC1 (PIPA528055, Thermo-Scientific, 3 μl of antibody in 20 μl of 2%BSA/1X PBS) during 30 min rotating at 4°C.

    Techniques: Multiple Displacement Amplification, Transmission Assay, Transmission Electron Microscopy, Flow Cytometry, Cytometry, Labeling, Negative Control