Team:Slovenia/Notebook

From 2012.igem.org

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<li>Purity and amount of DNA was determined using NanoDrop. </li>
<li>Purity and amount of DNA was determined using NanoDrop. </li>
</ol>
</ol>
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</br>
<h3>RESTRICTION DIGEST</h3>
<h3>RESTRICTION DIGEST</h3>
<ol>
<ol>
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</ol>
</ol>
</br>
</br>
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<p><b>PCR product purification </b></p>
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<p><b>PCR product purification</b>. Desired PCR products were purified by GeneJet Gel Extraction Kit according to the manufacturer's protocol. </p>
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<p>Desired PCR products were purified by GeneJet Gel Extraction Kit according to the manufacturer's protocol. </p>
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<p><b>DNA concentration. </b> An aliquot of the isolated DNA was analyzed using NanoDrop. </p>
<p><b>DNA concentration. </b> An aliquot of the isolated DNA was analyzed using NanoDrop. </p>
  <h3>Gibson assembly </h3>
  <h3>Gibson assembly </h3>
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<li>After incubation, the entire master mix volume was transformed into competent bacterial cells. </li>
<li>After incubation, the entire master mix volume was transformed into competent bacterial cells. </li>
</ol>
</ol>
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</br>
<h3>Ligation</h3>
<h3>Ligation</h3>
<p>T4 ligase ligates the 5' phosphate and the 3'-hydroxyl groups of DNA. </p>
<p>T4 ligase ligates the 5' phosphate and the 3'-hydroxyl groups of DNA. </p>
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<li>After incubation part of the ligation mixture was used for the transformation of bacterial cells (see: transformation of bacteria). </li>
<li>After incubation part of the ligation mixture was used for the transformation of bacterial cells (see: transformation of bacteria). </li>
</ol>
</ol>
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</br>
<h3> Culturing bacteria</h3>
<h3> Culturing bacteria</h3>
<p>For plasmid DNA propagation two bacterial strains were used: <b>DH5alpha</b> [<i>fhuA2Δ(argF-lacZ)U169 phoA glnV44 Φ80 Δ(lacZ)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17</i>] and <b>TOP10</b> [<i>mcrA, Δ(mrr-hsdRMS-mcrBC), Phi80lacZ(del)M15, ΔlacX74, deoR, recA1, araD139, Δ(ara-leu)7697, galU, galK, rpsL(SmR), endA1,nupG</i>]. </p>
<p>For plasmid DNA propagation two bacterial strains were used: <b>DH5alpha</b> [<i>fhuA2Δ(argF-lacZ)U169 phoA glnV44 Φ80 Δ(lacZ)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17</i>] and <b>TOP10</b> [<i>mcrA, Δ(mrr-hsdRMS-mcrBC), Phi80lacZ(del)M15, ΔlacX74, deoR, recA1, araD139, Δ(ara-leu)7697, galU, galK, rpsL(SmR), endA1,nupG</i>]. </p>
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<p><b> Luria Broth (LB) </b>: 10 g/L tryptone,    5 g/L yeast extract,    10 g/L NaCl,    media is supplemented with suitable antibiotics depending on the selection marker on the transfected plasmid: ampicilin 100 mg/L or kanamycin 50 mg/L.<p>
<p><b> Luria Broth (LB) </b>: 10 g/L tryptone,    5 g/L yeast extract,    10 g/L NaCl,    media is supplemented with suitable antibiotics depending on the selection marker on the transfected plasmid: ampicilin 100 mg/L or kanamycin 50 mg/L.<p>
<p><b> LB agar plates</b>: LB with 1.5% agar, media is supplemented with suitable antibiotics depending on the selection marker on the transfected plasmid.</p>
<p><b> LB agar plates</b>: LB with 1.5% agar, media is supplemented with suitable antibiotics depending on the selection marker on the transfected plasmid.</p>
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  <h3>Transformation of bacteria</h3>
  <h3>Transformation of bacteria</h3>
<p>  E. coli DH5alpha and TOP10 competent cells were used for the propagation of plasmid DNA. </p>
<p>  E. coli DH5alpha and TOP10 competent cells were used for the propagation of plasmid DNA. </p>
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<li>The selection for plasmid containing and therefore antibiotic resistant bacteria was conducted by plating them on antibiotic containing LB-agar plates. </li>
<li>The selection for plasmid containing and therefore antibiotic resistant bacteria was conducted by plating them on antibiotic containing LB-agar plates. </li>
</ol>
</ol>
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</br>
  <h3>Glycerol stock for long term storage of bacteria</h3>
  <h3>Glycerol stock for long term storage of bacteria</h3>
<ol>
<ol>
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<li>Afterwards the glycerol stock was stored at -80 °C. </li>
<li>Afterwards the glycerol stock was stored at -80 °C. </li>
</ol>
</ol>
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  <h2> Cell cultures </h2>
  <h2> Cell cultures </h2>
<h3>Eucaryotic cell lines and cultivation</h3>
<h3>Eucaryotic cell lines and cultivation</h3>
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<p><b>HEK293T</b> cell line is derived form HEK293 cells. HEK293T cells express the SV40 large T-antigen that enables episomal replication of plasmids containing the SV40 origin of replication in transfected cells. Cells were grown in DMEM medium supplemented with 10% FBS. </p>
<p><b>HEK293T</b> cell line is derived form HEK293 cells. HEK293T cells express the SV40 large T-antigen that enables episomal replication of plasmids containing the SV40 origin of replication in transfected cells. Cells were grown in DMEM medium supplemented with 10% FBS. </p>
<p><b>NK-92</b> is an interleukin-2 (IL-2) dependent natural killer cell line derived from peripheral blood mononuclear cells from patient with non-Hodgkin's lymphoma. The cell line is cytotoxic to a wide range of malignant cells. Cells were grown in RPMI medium supplemented with 20% FBS and 100 U/ml IL-2.</p>
<p><b>NK-92</b> is an interleukin-2 (IL-2) dependent natural killer cell line derived from peripheral blood mononuclear cells from patient with non-Hodgkin's lymphoma. The cell line is cytotoxic to a wide range of malignant cells. Cells were grown in RPMI medium supplemented with 20% FBS and 100 U/ml IL-2.</p>
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</br>
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<h3>Subculturing monolayer cell cultures</h3>
<h3>Subculturing monolayer cell cultures</h3>
<ol>
<ol>
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<li>Return the cells in a T-75 flask to the incubator (37 °C, 5 % CO2). </li>
<li>Return the cells in a T-75 flask to the incubator (37 °C, 5 % CO2). </li>
</ol>
</ol>
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<p><b>Cell plating</b></p>
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</br>
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<h3>Cell plating</h3>
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<ol>
<ol>
<li>Count cells. </li>
<li>Count cells. </li>
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<li>Transfer the cells into an appropriate plate and place in a cell culture incubator. </li>
<li>Transfer the cells into an appropriate plate and place in a cell culture incubator. </li>
</ol>
</ol>
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<p><b>media and buffers</b></p>
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<p><b>Media and buffers</b></p>
<p><b>DMEM</b> supplemented with:    1 % L-Glutamine (GlutaMax),    10 % FBS,    Optionally: 1% Pen/Strep.</p>
<p><b>DMEM</b> supplemented with:    1 % L-Glutamine (GlutaMax),    10 % FBS,    Optionally: 1% Pen/Strep.</p>
<p><b>RPMI</b> supplemented with:    1 % L-Glutamine (GlutaMax),    20 % FBS.</p>
<p><b>RPMI</b> supplemented with:    1 % L-Glutamine (GlutaMax),    20 % FBS.</p>
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</br>
<h3>Induction of cells</h3>
<h3>Induction of cells</h3>
<ol>
<ol>
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<li>After 24 h of stimulation, cells were lysed and NF-κB activation was assessed using dual luciferase assay. </li>
<li>After 24 h of stimulation, cells were lysed and NF-κB activation was assessed using dual luciferase assay. </li>
</ol>
</ol>
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</br>
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<h3> Biological assay-IFN-alpha </h3>
<h3> Biological assay-IFN-alpha </h3>
<ol>
<ol>
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<li>After 24 hours of incubation, dual luciferase reporter assay was preformed. </li>
<li>After 24 hours of incubation, dual luciferase reporter assay was preformed. </li>
</ol>
</ol>
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</br>
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<h3>ELISA for IFN-alpha</h3>
<h3>ELISA for IFN-alpha</h3>
<ol>
<ol>
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<li>Supernatants were collected after 16h and serial dilutions were measured for IFN-alpha levels by Human IFN-alpha Instant Elisa (eBioscience). </li>
<li>Supernatants were collected after 16h and serial dilutions were measured for IFN-alpha levels by Human IFN-alpha Instant Elisa (eBioscience). </li>
</ol>
</ol>
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</br>
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<h3>Plate reader-fluorescence(The Switch) </h3>  
<h3>Plate reader-fluorescence(The Switch) </h3>  
<ol>
<ol>
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<li>Fluorescence was measured using an automated plate reader. </li>
<li>Fluorescence was measured using an automated plate reader. </li>
</ol>
</ol>
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</br>
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<h3>Plate reader-luminescence  (The Switch) </h3>  
<h3>Plate reader-luminescence  (The Switch) </h3>  
<ol>
<ol>
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<li>Luminescence of expressed reporter firefly luciferase was measured with Orion (Berthold Technologies) using Luciferase buffer with luciferin as a substrate. For normalization Renilla luciferase activity was used. The Renilla luciferase was measured using Renilla buffer supplemented with coelenterazine. </li>
<li>Luminescence of expressed reporter firefly luciferase was measured with Orion (Berthold Technologies) using Luciferase buffer with luciferin as a substrate. For normalization Renilla luciferase activity was used. The Renilla luciferase was measured using Renilla buffer supplemented with coelenterazine. </li>
</ol>
</ol>
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</br>
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<h2>Microscopy </h2>
<h2>Microscopy </h2>
<p>For spatial and temporal imaging of samples a Leica TCS SP5 laser scanning microscope mounted on a Leica DMI 6000 CS inverted microscope (Leica Microsystems, Germany) with a 10× and 20× dry objective and an HCX plan apo 63× oil (NA 1.4) oil immersion objective was used. For image analysis we used ImageJ (Image Processing and Analysis in Java) software (http://rsbweb.nih.gov/ij/) measuring the mean grey values of each cell containing the promoter of interest. </p>
<p>For spatial and temporal imaging of samples a Leica TCS SP5 laser scanning microscope mounted on a Leica DMI 6000 CS inverted microscope (Leica Microsystems, Germany) with a 10× and 20× dry objective and an HCX plan apo 63× oil (NA 1.4) oil immersion objective was used. For image analysis we used ImageJ (Image Processing and Analysis in Java) software (http://rsbweb.nih.gov/ij/) measuring the mean grey values of each cell containing the promoter of interest. </p>
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</br>
<h3>Microscopy-cell viability with Hoechst and SytoxGreen514 (Safety mechanisms) </h3>
<h3>Microscopy-cell viability with Hoechst and SytoxGreen514 (Safety mechanisms) </h3>
<p><b>Hoechst</b> dye is a membrane permeable dye and stains all cells in a culture. On the other hand a <b>SytoxGreen514</b> dye is a membrane impermeable dye staining only dead cells. Both dyes, blue fluorescent Hoechst and green fluorescent SytoxGreen514, bind to nucleic acids causing emission of fluorescent light. </p>
<p><b>Hoechst</b> dye is a membrane permeable dye and stains all cells in a culture. On the other hand a <b>SytoxGreen514</b> dye is a membrane impermeable dye staining only dead cells. Both dyes, blue fluorescent Hoechst and green fluorescent SytoxGreen514, bind to nucleic acids causing emission of fluorescent light. </p>
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<li>A 405-nm diode laser was used to excite Hoechst and a 514-nm line of 25 mW multi ion argon laser was used to excite SytoxGreen514. Successive images excited at 405 and 514 nm were captured. Fluorescence emission was detected at 450-500 nm and 520-560 nm for Hoechst and SytoxGreen respectively. </li>
<li>A 405-nm diode laser was used to excite Hoechst and a 514-nm line of 25 mW multi ion argon laser was used to excite SytoxGreen514. Successive images excited at 405 and 514 nm were captured. Fluorescence emission was detected at 450-500 nm and 520-560 nm for Hoechst and SytoxGreen respectively. </li>
</ol>
</ol>
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</br>
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<h3>Microscopy-cell growth (Safety mechanisms) </h3>
<h3>Microscopy-cell growth (Safety mechanisms) </h3>
<ol>
<ol>
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<li>A 514-nm line of 25 mW multi ion argon laser was used to excite mCitrine reporter protein. Fluorescence emission was detected at 520-560 nm for mCitrine. Bright field images were used to visualize the number of cells. </li>
<li>A 514-nm line of 25 mW multi ion argon laser was used to excite mCitrine reporter protein. Fluorescence emission was detected at 520-560 nm for mCitrine. Bright field images were used to visualize the number of cells. </li>
</ol>
</ol>
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</br>
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<h3>Microscopy-cell count (Safety mechanisms) </h3>
<h3>Microscopy-cell count (Safety mechanisms) </h3>
<ol>
<ol>
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<li>Viable cell number was determined by counting the cells under a light microscope using a Bürker-Türk counting chamber. </li>
<li>Viable cell number was determined by counting the cells under a light microscope using a Bürker-Türk counting chamber. </li>
</ol>
</ol>
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</br>
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<h3>Microscopy-detection of reporter proteins (The Switch) </h3>
<h3>Microscopy-detection of reporter proteins (The Switch) </h3>
<p>Fluorescent proteins were used as reporters in "The switch experiments". The fluorescent proteins used were blue (tagBFP), yellow (mCitrine), orange (mCherry) and red (mNeptun) fluorescent proteins. mCherry was used as transfection control while the others were used as reporters of "the switch".</p>
<p>Fluorescent proteins were used as reporters in "The switch experiments". The fluorescent proteins used were blue (tagBFP), yellow (mCitrine), orange (mCherry) and red (mNeptun) fluorescent proteins. mCherry was used as transfection control while the others were used as reporters of "the switch".</p>
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<li>A 405-nm diode laser was used to excite tagBFP, a 514-nm line of 25 mW multi-ion argon laser was used for mCitirne, a 543-nm HeNe laser was used for mCherry and a 633-nm HeNe laser was used to excite mNeptune. Successive images excited at 405, 514, 543 and 633 nm were captured. All intensities of laser and photomultipliers were kept unchanged during one set of experiments to enable comparison of images. Fluorescence emission was detected at 450-500 nm, 520-560 nm, 560-600 nm and 640-700 nm for tagBFP, mCitrine, mCherry and mNeptune, SytoxGreen respectively. </li>
<li>A 405-nm diode laser was used to excite tagBFP, a 514-nm line of 25 mW multi-ion argon laser was used for mCitirne, a 543-nm HeNe laser was used for mCherry and a 633-nm HeNe laser was used to excite mNeptune. Successive images excited at 405, 514, 543 and 633 nm were captured. All intensities of laser and photomultipliers were kept unchanged during one set of experiments to enable comparison of images. Fluorescence emission was detected at 450-500 nm, 520-560 nm, 560-600 nm and 640-700 nm for tagBFP, mCitrine, mCherry and mNeptune, SytoxGreen respectively. </li>
</ol>
</ol>
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</br>
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<h3>Microscopy-alginate degradation (Microencapsulation) </h3>
<h3>Microscopy-alginate degradation (Microencapsulation) </h3>
<p>To observe the degradation of alginate beads, 2000 kDa FITC-dexstran (Sigma) was added to 200 µL of culture medium containing alginate beads with immobilized HEK 293T cells. Because FITC-dexstran cannot penetrate the alginate beads, we can easily observe bead degradation uppon addition of alginate lyase from Sphingobacterium multivorum (Sigma).</p>
<p>To observe the degradation of alginate beads, 2000 kDa FITC-dexstran (Sigma) was added to 200 µL of culture medium containing alginate beads with immobilized HEK 293T cells. Because FITC-dexstran cannot penetrate the alginate beads, we can easily observe bead degradation uppon addition of alginate lyase from Sphingobacterium multivorum (Sigma).</p>
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<li>A 488-nm line of 25 mW multi-ion argon laser was used for FITC. Fluorescence emission was detected at 520-560 nm. At the same time a bright field image was taken. </li>
<li>A 488-nm line of 25 mW multi-ion argon laser was used for FITC. Fluorescence emission was detected at 520-560 nm. At the same time a bright field image was taken. </li>
</ol>
</ol>
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</br>
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<h3>Microscopy-encapsulated cell viability (Microencapsulation) </h3>
<h3>Microscopy-encapsulated cell viability (Microencapsulation) </h3>
<p>To observe encapsulated cells' viability, HEK 293T cells were stained with Hoechst and 7-aminoactinomycin D (7-AAD) viability stains. Hoechst stains both living and dead cells, while 7-AAD stains dead cells only.</p>
<p>To observe encapsulated cells' viability, HEK 293T cells were stained with Hoechst and 7-aminoactinomycin D (7-AAD) viability stains. Hoechst stains both living and dead cells, while 7-AAD stains dead cells only.</p>
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<li>Fluorescence emission was detected at 450-500 nm and 600-700 nm for Hoechst and 7-AAD respectively. </li>
<li>Fluorescence emission was detected at 450-500 nm and 600-700 nm for Hoechst and 7-AAD respectively. </li>
</ol>
</ol>
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</br>
<h2>Flow cytometry </h2>
<h2>Flow cytometry </h2>
<p>Flow cytometry is a laser based technology employed in cell counting and biomarker detection. It allows simultaneous multiparametric analysis of the physical as well as biochemical and biological characteristics of particles. We used a CyFlow Space (Partec) flow cytometer equipped with three lasers (405, 488 and 633 nm). The CyFlow detects forward scatter and side scatter signals and up to 6 colors of fluorescence.</p>
<p>Flow cytometry is a laser based technology employed in cell counting and biomarker detection. It allows simultaneous multiparametric analysis of the physical as well as biochemical and biological characteristics of particles. We used a CyFlow Space (Partec) flow cytometer equipped with three lasers (405, 488 and 633 nm). The CyFlow detects forward scatter and side scatter signals and up to 6 colors of fluorescence.</p>
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</br>
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<h3>Flow cytometry - the annexin assay (Safety mechanisms) </h3>
<h3>Flow cytometry - the annexin assay (Safety mechanisms) </h3>
<p>To determine the percentage of cells undergoing apoptosis as a result of herpes simplex virus thymidine kinase (HSV-TK) (pCMV-mGMK_TK30) transfection and ganciclovir treatment we labelled cells with Annexin V conjugated with phycoerythrin (PE). Annexin V is a Ca2+ dependent phospholipid-binding protein that has a high affinity for the phospholipid phosphatidylserine and therefore binds to apoptotic cells with  phosphatidylserine exposed on their surface.</p>
<p>To determine the percentage of cells undergoing apoptosis as a result of herpes simplex virus thymidine kinase (HSV-TK) (pCMV-mGMK_TK30) transfection and ganciclovir treatment we labelled cells with Annexin V conjugated with phycoerythrin (PE). Annexin V is a Ca2+ dependent phospholipid-binding protein that has a high affinity for the phospholipid phosphatidylserine and therefore binds to apoptotic cells with  phosphatidylserine exposed on their surface.</p>
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<li>Along with site and forward scatter, the signal in the FL2 channel  (540-580 nm) was also recorded. </li>
<li>Along with site and forward scatter, the signal in the FL2 channel  (540-580 nm) was also recorded. </li>
</ol>
</ol>
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</br>
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<h3>Flow cytometry - the propidium iodide assay (Safety mechanisms) </h3>
<h3>Flow cytometry - the propidium iodide assay (Safety mechanisms) </h3>
<p>To determine the percentage of dead cells due to cytotoxic activity of natural killer cells against HEK293T cells expressing MICA protein, cells were stained with propidium iodide dye, which intercalates into DNA and stains only dead cells, because it is a membrane impermeant fluorescent molecule.</p>
<p>To determine the percentage of dead cells due to cytotoxic activity of natural killer cells against HEK293T cells expressing MICA protein, cells were stained with propidium iodide dye, which intercalates into DNA and stains only dead cells, because it is a membrane impermeant fluorescent molecule.</p>
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<li>Along with site and forward scatter the signal in the FL1 channel (530-580 nm) was also recorded. </li>
<li>Along with site and forward scatter the signal in the FL1 channel (530-580 nm) was also recorded. </li>
</ol>
</ol>
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</br>
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<h3>Flow cytometry - detection of reporter proteins (The Switch) </h3>
<h3>Flow cytometry - detection of reporter proteins (The Switch) </h3>
<p>Reporters such as fluorescent proteins were used to detect the expression of effectors in "The switch experiments". As reporters we used blue (tagBFP) and yellow (mCitrine) fluorescent proteins.</p>
<p>Reporters such as fluorescent proteins were used to detect the expression of effectors in "The switch experiments". As reporters we used blue (tagBFP) and yellow (mCitrine) fluorescent proteins.</p>
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<li>Along with site and forward scatter signals in the FL1 (540-580 nm) channel (mCitrine) and the FL5 (450-480 nm) channel (tagBFP) were also recorded. </li>
<li>Along with site and forward scatter signals in the FL1 (540-580 nm) channel (mCitrine) and the FL5 (450-480 nm) channel (tagBFP) were also recorded. </li>
</ol>
</ol>
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</br>
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<h2>Microencapsulation </h2>
<h2>Microencapsulation </h2>
<h3>Cell preparation for encapsulation</h3>
<h3>Cell preparation for encapsulation</h3>
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<li>10 mL of pre-warmed alginate solution (1,5%) was added to cell suspension. </li>
<li>10 mL of pre-warmed alginate solution (1,5%) was added to cell suspension. </li>
</ol>
</ol>
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</br>
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<h3>Encapsulation</h3>
<h3>Encapsulation</h3>
<ol>
<ol>
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<p><b>0,03% alginate</b> in MOPS buffer (pH = 7,2) </p>
<p><b>0,03% alginate</b> in MOPS buffer (pH = 7,2) </p>
<p><b>1,5% alginate</b> (low viscosity) in MOPS buffer (pH = 7,2) </p>
<p><b>1,5% alginate</b> (low viscosity) in MOPS buffer (pH = 7,2) </p>
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</br>
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<h2>Protein detection</h2>
<h2>Protein detection</h2>
<h3>SDS-PAGE</h3>
<h3>SDS-PAGE</h3>
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<li>The membrane was washed with MQ and PBS and blocked for 1,5 h by incubation in I-Block blocking reagent at room temperature. </li>
<li>The membrane was washed with MQ and PBS and blocked for 1,5 h by incubation in I-Block blocking reagent at room temperature. </li>
</ol>
</ol>
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</br>
<h3>Immunodetection </h3>
<h3>Immunodetection </h3>

Revision as of 18:47, 26 September 2012