Team:Slovenia/Notebook

From 2012.igem.org

(Difference between revisions)
Line 988: Line 988:
<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>
<ol>
<ol>
-
<li>HEK293 cells were seeded in an 8-well microscope chamber and transfected with 100 ng CMV-mGMK_TK30 (pPCMV_mGMK:TK30). </li>
+
<li>HEK293 cells were seeded in an 8-well microscope chamber and transfected with 200 ng CMV-mGMK_TK30 (pPCMV_mGMK:TK30). </li>
<li>Ganciclovir (GCV) in concentrations 0, 10 and 100 μg/mL was added to the cell cultures. </li>
<li>Ganciclovir (GCV) in concentrations 0, 10 and 100 μg/mL was added to the cell cultures. </li>
<li>After 5 days of cultivation, a Hoechst dye (0.4 μg/mL) and a SytoxGreen514 dye (1 μM) were used to stain cells and discriminate between live and dead cells. </li>
<li>After 5 days of cultivation, a Hoechst dye (0.4 μg/mL) and a SytoxGreen514 dye (1 μM) were used to stain cells and discriminate between live and dead cells. </li>
<li>Cells were incubated for approximately 10 minutes in the dark at 37 °C before imaging. </li>
<li>Cells were incubated for approximately 10 minutes in the dark at 37 °C before imaging. </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>
+
<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 nm 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>
</br>
</br>
Line 998: Line 998:
<h3>Microscopy-cell growth (Safety mechanisms) </h3>
<h3>Microscopy-cell growth (Safety mechanisms) </h3>
<ol>
<ol>
-
<li>HEK293 cells were seeded in an 8-well microscope chamber and transfected with 100 ng mGMK:TK30 (pPCMV_mGMK:TK30) and/or 20 ng mCitirne (pPCMV-mCitrine) (for transfection control). </li>
+
<li>HEK293 cells were seeded in an 8-well microscope chamber and transfected with 100 ng mGMK:TK30 (pPCMV_mGMK:TK30) and/or 20 ng GFP (pPCMV-GFP) (for transfection control). </li>
<li>Ganciclovir (GCV) in concentrations 0, 10 and 100 μg/mL was added to the cell cultures. </li>
<li>Ganciclovir (GCV) in concentrations 0, 10 and 100 μg/mL was added to the cell cultures. </li>
<li>After 5 days of cultivation, a cell cultures were imaged. </li>
<li>After 5 days of cultivation, a cell cultures were imaged. </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>
+
<li>A 514-nm line of 25 mW multi ion argon laser was used to excite GFP reporter protein. Fluorescence emission was detected at 520-560 nm for GFP. Bright field images were used to visualize the number of cells. </li>
</ol>
</ol>
</br>
</br>
Line 1,007: Line 1,007:
<h3>Microscopy-cell count (Safety mechanisms) </h3>
<h3>Microscopy-cell count (Safety mechanisms) </h3>
<ol>
<ol>
-
<li>HEK293 cells were seeded in 12-well plates and transfected with 100 ng mGMK:TK30 (pPCMV_mGMK:TK30). </li>
+
<li>HEK293 cells were seeded in 12-well plates and transfected with different amounts of mGMK:TK30 (pPCMV_mGMK:TK30) as indicated in Figure legend. </li>
-
<li>Cell cultures were treated with ganciclovir (GCV) in concentrations as indicated in the figure legend. </li>
+
<li>Cell cultures were treated with ganciclovir (GCV) in concentrations as indicated in the Figure legend. </li>
<li>After incubation the cells were resuspended by pipetting. </li>
<li>After incubation the cells were resuspended by pipetting. </li>
<li>Cells suspensions were then mixed with trypan blue. </li>
<li>Cells suspensions were then mixed with trypan blue. </li>
Line 1,027: Line 1,027:
<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 upon addition of alginate lyase from Sphingobacterium multivorum (Sigma).</p>
<ol>
<ol>
<li>Alginate beads suspended in culture medium were seeded in an 8-well microscope chamber (200 µL). </li>
<li>Alginate beads suspended in culture medium were seeded in an 8-well microscope chamber (200 µL). </li>
Line 1,035: Line 1,035:
<li>Screenshots were collected for at least 15 minutes. </li>
<li>Screenshots were collected for at least 15 minutes. </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>
<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>
 +
</br>
 +
 +
 +
<h3>Microscopy-secreted alginate lyase enzymatic activity (Microencapsulation)</h3>
 +
<ol>
 +
<li>HEK293T cells were seeded 1×10<sup>6</sup> on 10 cm cell culture dish and grown in DMEM medium supplemented with 10 % FBS.</li>
 +
<li>After reaching 50 – 70 % confluency, cells were transfected with 15 μg of DNA per culture dish with jetPEI transfection reagent (Polyplus Transfection).</li>
 +
<li>Protein production lasted for 72 hours.</li>
 +
<li>Cell supernatants were collected and concentrated 50-times using Sartorius Vivaspin 6 concentrators.</li>
 +
<li>Alginate beads were produced with Büchi BIOTECH Encapsulator (see Microencapsulation: Encapsulation procedure 1.-6.).</li>
 +
<li>Beads were incubated with concentrated supernatants for 72 hours in an 8-well microscope chamber.</li>
 +
<li>20 µL of 1 mg/mL FITC-dextran were added into wells.</li>
 +
<li>A 488-nm line of 25 mW multi-ion argon laser was used for  FITC detection. Fluorescence emission was detected at 520-560 nm.</li>
 +
<li>Beads' diameters were assessed using Leica LAS Image Analysis software.</li>
</ol>
</ol>
</br>
</br>

Revision as of 16:59, 26 October 2012