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| METHODS | | METHODS |
| <ol> | | <ol> |
− | <b>Preparation</b> | + | <i>Preparation</i> |
| <li> Warm medium (DMEM (1x) + GlutaMAXTM-I + 10% FBS), PBS and TE solution in a water bath @ 37ºC</li> | | <li> Warm medium (DMEM (1x) + GlutaMAXTM-I + 10% FBS), PBS and TE solution in a water bath @ 37ºC</li> |
| <li> Spray tubes, bottles, racks and everything that goes inside the hood with 70% ethanol</li> | | <li> Spray tubes, bottles, racks and everything that goes inside the hood with 70% ethanol</li> |
− | <b>Taking off the medium</b> | + | <i>Taking off the medium</i> |
| <li>Take off almost all the medium inside the flask</li> | | <li>Take off almost all the medium inside the flask</li> |
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− | <b>Washing the cells</b> | + | <i>Washing the cells</i> |
| <li>Wash gently the cells with PBS</li> | | <li>Wash gently the cells with PBS</li> |
− | <p><b>Trypsinization</b> | + | <p><i>Trypsinization</i> |
| <li>Cover the flask with 3-5 mL with Trypsin/EDTA solution</li> | | <li>Cover the flask with 3-5 mL with Trypsin/EDTA solution</li> |
| <li> Incubate for 3 - 5 minutes @ 37ºC</li> | | <li> Incubate for 3 - 5 minutes @ 37ºC</li> |
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| <li> Add 5 mL medium to inactivate trypsin</li> | | <li> Add 5 mL medium to inactivate trypsin</li> |
| <li>Centrifuge 400 x g for 5 minutes @ 21 ºC (Eppendorf 5810 R)</li></p> | | <li>Centrifuge 400 x g for 5 minutes @ 21 ºC (Eppendorf 5810 R)</li></p> |
− | <b>Adding new medium</b> | + | <i>Adding new medium</i> |
| <li>Remove medium + TE without touching the pellet</li> | | <li>Remove medium + TE without touching the pellet</li> |
| <li> Add 5 mL of medium to the centrifuge tube with cells</li> | | <li> Add 5 mL of medium to the centrifuge tube with cells</li> |
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| <li>Cells were pipetted into a 96 well plate with cell densities reducing by half in each following column (8 replicates)</li> | | <li>Cells were pipetted into a 96 well plate with cell densities reducing by half in each following column (8 replicates)</li> |
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− | <b>Cell fixation</b><br> | + | <i>Cell fixation</i><br> |
| <li> Fix the cells in 4% paraformaldehyde for 15 min @ RT<br></li> | | <li> Fix the cells in 4% paraformaldehyde for 15 min @ RT<br></li> |
| <li> Wash with PBS three times (3 times, 5 minutes each)</li> | | <li> Wash with PBS three times (3 times, 5 minutes each)</li> |
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− | <p><b>Cell permeabilization for intracellular biomarkers</b> | + | <p><i>Cell permeabilization for intracellular biomarkers</i> |
| <li>Incubate with PBS + 0.1% Triton X-100 for 10 minutes @ RT</li> | | <li>Incubate with PBS + 0.1% Triton X-100 for 10 minutes @ RT</li> |
| <li>Wash with PBS three times (5 minutes each)</li></p> | | <li>Wash with PBS three times (5 minutes each)</li></p> |
− | <p><b>Blocking</b> | + | <p><i>Blocking</i> |
| <li> Block with PBS + 3% BSA for 1 hour @ RT</li> | | <li> Block with PBS + 3% BSA for 1 hour @ RT</li> |
| <li> Wash with PBS (3 times, 5 minutes each)</li></p> | | <li> Wash with PBS (3 times, 5 minutes each)</li></p> |
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| <li>Incubate the cells with primary antibody (1:200 – 1:2000) in PBS + 1% BSA for 3 hours @ RT</li> | | <li>Incubate the cells with primary antibody (1:200 – 1:2000) in PBS + 1% BSA for 3 hours @ RT</li> |
| <li>Wash with PBS (3 times, 5 minutes each)</li></p>--> | | <li>Wash with PBS (3 times, 5 minutes each)</li></p>--> |
− | <p><b>DAPI/Hoechst addition</b> | + | <p><i>DAPI/Hoechst addition</i> |
| <li>Incubate the cells with secondary antibody (1:200) in PBS + 1% BSA for 1 hour @ RT</li> | | <li>Incubate the cells with secondary antibody (1:200) in PBS + 1% BSA for 1 hour @ RT</li> |
| <li>Wash with PBS (3 times, 5 minutes each)</li> | | <li>Wash with PBS (3 times, 5 minutes each)</li> |
| <li> Incubate the cells on 1:3000 DAPI or Hoechst for 5 minutes @ 4 ºC</li> | | <li> Incubate the cells on 1:3000 DAPI or Hoechst for 5 minutes @ 4 ºC</li> |
| <li> Wash with PBS (3 times, 5 minutes each)</li></p> | | <li> Wash with PBS (3 times, 5 minutes each)</li></p> |
− | <p><b>Imaging</b> | + | <p><i>Imaging</i> |
| <li>Take pictures in a confocal microscope according to the desired channel (bright field, DAPI, Texas Red, GFP) at 4x, 10x, 20x</li></p> | | <li>Take pictures in a confocal microscope according to the desired channel (bright field, DAPI, Texas Red, GFP) at 4x, 10x, 20x</li></p> |
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