<p><span>Aim: to build a plasmid (pENTR1A-PSMA-GFP) that allows us to detect PSMA-positive prostate cancer cells by measuring the fluorescence by GFP.</span></p>
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<aname = "build1"></a>
<h5><span>Build</span></h5>
<p><span>We purchased the pENTR1A backbone and another plasmid containing the PSMA promoter and GFP gene. Both plasmids were digested and gel electrophoresis was performed.</span></p>
<p><span>We then purified the agarose gel and the two genes were ligated to form plasmid pENTR1A-PSMA-GFP. (for more information refer to</span><ahref = "#cycle3"> cycle 3</a><span>)</span></p>
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<p><span>After the ligated plasmids were extracted, we transfected different concentrations of them into multiple concentrations of PSMA-positive cancer cell line MLLB-2.</span></p>
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<h5><span>Test</span></h5>
<p><span>Fluorescence given out by GFP was visualised by using an inverted microscope with a live cell-monitoring system.</span></p>
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<aname = "desgin4"></a>
<aname = "design4"></a>
<h5><span>Design</span></h5>
<p><span>We followed a set of standard protocols, found in manufacturers’ product sheets, during our process of plasmid construction.</span></p>
<listyle="font-weight: 400;"><strong>Add </strong><spanstyle="font-weight: 400;">20 μL/well of </span><strong>medium</strong><spanstyle="font-weight: 400;"> from the transfected cells </span><strong>within</strong><strong>72 hours</strong><spanstyle="font-weight: 400;"> after transfection</span><spanstyle="font-weight: 400;">to a black opaque 96-well plate. (For Transfection Protocol, please refer 3.4 Transfection)</span></li>
<li><spanstyle="font-weight: 400;">Add 50 μL of </span><strong>coelenterazine working solution</strong><spanstyle="font-weight: 400;"> to each well and </span><strong>detect the light output </strong><spanstyle="font-weight: 400;">immediately</span></li>
<li><strong>Add </strong><span>20 μL/well of </span><strong>medium</strong><span> from the transfected cells </span><strong>within</strong><strong>72 hours</strong><span> after transfection</span><span>to a black opaque 96-well plate. (For Transfection Protocol, please refer 3.4 Transfection)</span></li>
<li><span>Add 50 μL of </span><strong>coelenterazine working solution</strong><span> to each well and </span><strong>detect the light output </strong><span>immediately</span></li>
<listyle="font-weight: 400;"><spanstyle="font-weight: 400;">Remove original culture medium and add 100 µL of fresh medium</span></li>
<listyle="font-weight: 400;"><spanstyle="font-weight: 400;">Add 10 µL of the 12-mM MTT solution to each well. Include a negative control by adding 10 µL of the MTT solution to 100 µL of medium alone.</span></li>
<listyle="font-weight: 400;"><spanstyle="font-weight: 400;">Incubate at 37°C for 2–5 hours. </span></li>
<listyle="font-weight: 400;"><spanstyle="font-weight: 400;">Remove 85 µL of medium from the wells.</span></li>
<listyle="font-weight: 400;"><spanstyle="font-weight: 400;">Add 50 µL of DMSO to each well, then pipet up and down thoroughly to mix.</span></li>
<listyle="font-weight: 400;"><spanstyle="font-weight: 400;">Incubate at 37°C for 10 minutes. </span></li>
<listyle="font-weight: 400;"><spanstyle="font-weight: 400;">Pipet up and down to mix each sample again, then read the absorbance at 540 nm. </span></li>
<li><span>Remove original culture medium and add 100 µL of fresh medium</span></li>
<li><span>Add 10 µL of the 12-mM MTT solution to each well. Include a negative control by adding 10 µL of the MTT solution to 100 µL of medium alone.</span></li>
<li><span>Incubate at 37°C for 2–5 hours.</span></li>
<li><span>Remove 85 µL of medium from the wells.</span></li>
<li><span>Add 50 µL of DMSO to each well, then pipet up and down thoroughly to mix.</span></li>
<li><span>Incubate at 37°C for 10 minutes.</span></li>
<li><span>Pipet up and down to mix each sample again, then read the absorbance at 540 nm.</span></li>
<listyle="font-weight: 400;"><strong>Dilute</strong><spanstyle="font-weight: 400;"> the poly-D-lysine solution with </span><strong>sterile DPBS</strong><spanstyle="font-weight: 400;"> to prepare a 50 μg/mL working solution</span></li>
<listyle="font-weight: 400;"><strong>Coat</strong><spanstyle="font-weight: 400;"> the surface of 75 cm</span><spanstyle="font-weight: 400;">2</span><spanstyle="font-weight: 400;"> culture vessel with 11.7 mL </span><strong>working solution</strong><spanstyle="font-weight: 400;"> of poly-D-lysine</span></li>
<listyle="font-weight: 400;"><strong>Incubate</strong><spanstyle="font-weight: 400;"> the vessel at </span><strong>room temperature</strong><spanstyle="font-weight: 400;"> for an hour then </span><strong>remove</strong><spanstyle="font-weight: 400;"> the poly-D-lysine solution with a pipette gun.</span></li>
<listyle="font-weight: 400;"><strong>Rinse</strong><spanstyle="font-weight: 400;"> culture surface for 3 times with 24 mL distilled water and remove it from the flask.</span></li>
<listyle="font-weight: 400;"><spanstyle="font-weight: 400;">Leave the coated culture vessel uncovered in the biosafety cabinet to </span><strong>dry</strong><spanstyle="font-weight: 400;"> for </span><strong>2 hours</strong><spanstyle="font-weight: 400;">. Afterwards, the flask can be used immediately or store at </span><strong>4°C</strong></li>
<listyle="font-weight: 400;"><strong>Dilute</strong><spanstyle="font-weight: 400;"> the thawed laminin solution to 3 μg/mL with </span><strong>sterile distilled water</strong><spanstyle="font-weight: 400;">.</span></li>
<listyle="font-weight: 400;"><spanstyle="font-weight: 400;">Add </span><strong>laminin solution</strong><spanstyle="font-weight: 400;"> into poly-D-lysine coated culture vessel to cover the whole surface.</span></li>
<listyle="font-weight: 400;"><strong>Incubate</strong><spanstyle="font-weight: 400;"> and store in a </span><strong>37°C 5% CO</strong><strong>2</strong><strong> incubator</strong><spanstyle="font-weight: 400;">.</span></li>
<li><strong> Aspirate</strong><spanstyle="font-weight: 400;"> laminin solution from coated culture vessel and let it </span><strong>dry</strong><spanstyle="font-weight: 400;"> right before seeding cells.</span></li>
<li><strong>Dilute</strong><span> the poly-D-lysine solution with </span><strong>sterile DPBS</strong><span> to prepare a 50 μg/mL working solution</span></li>
<li><strong>Coat</strong><span> the surface of 75 cm</span><span>2</span><span> culture vessel with 11.7 mL </span><strong>working solution</strong><span> of poly-D-lysine</span></li>
<li><strong>Incubate</strong><span> the vessel at </span><strong>room temperature</strong><span> for an hour then </span><strong>remove</strong><span> the poly-D-lysine solution with a pipette gun.</span></li>
<li><strong>Rinse</strong><span> culture surface for 3 times with 24 mL distilled water and remove it from the flask.</span></li>
<li><span>Leave the coated culture vessel uncovered in the biosafety cabinet to </span><strong>dry</strong><span> for </span><strong>2 hours</strong><span>. Afterwards, the flask can be used immediately or store at </span><strong>4°C</strong></li>
<li><strong>Dilute</strong><span> the thawed laminin solution to 3 μg/mL with </span><strong>sterile distilled water</strong><span>.</span></li>
<li><span>Add </span><strong>laminin solution</strong><span> into poly-D-lysine coated culture vessel to cover the whole surface.</span></li>
<li><strong>Incubate</strong><span> and store in a </span><strong>37°C 5% CO</strong><strong>2</strong><strong> incubator</strong><span>.</span></li>
<li><strong>Aspirate</strong><span> laminin solution from coated culture vessel and let it </span><strong>dry</strong><span> right before seeding cells.</span></li>
