Difference between revisions of "Team:Paris Saclay/Notebook/October/19"
(→Wednesday 19th October) |
(→Cytometry measurements=) |
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| Line 5: | Line 5: | ||
===Visualization=== | ===Visualization=== | ||
| − | ===Cytometry measurements | + | ===Cytometry measurements=== |
''By Sylvie'' | ''By Sylvie'' | ||
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This preparation was divided in 4 tubes of 4ml and a falcon of 32 ml. | This preparation was divided in 4 tubes of 4ml and a falcon of 32 ml. | ||
| − | In | + | In the 4 tubes we added rapalog in order to test the fluorescence : |
| − | + | *tube 1: control without rapalog | |
| − | tube | + | *tube 2: 5 nM of rapalog |
| − | tube | + | *tube 3: 50 nM of rapalog |
| − | + | *tube 4: 500 nM of rrapalog | |
After 5 hours of culture, cytometry didn't show any fluorescence. | After 5 hours of culture, cytometry didn't show any fluorescence. | ||
| Line 29: | Line 29: | ||
For the falcon of the remaining preparation (32ml) : | For the falcon of the remaining preparation (32ml) : | ||
*Centrifuge | *Centrifuge | ||
| − | * Discard the supernatant | + | *Discard the supernatant |
*Resuspend the pellet in 15 ml of 100mM Tris (pH= 7,4), 100mM NaCl, 10 % glycerol | *Resuspend the pellet in 15 ml of 100mM Tris (pH= 7,4), 100mM NaCl, 10 % glycerol | ||
*Discardthe supernatant | *Discardthe supernatant | ||
*Resuspend in 800 μL of TNG buffer and 20μL of PMSI | *Resuspend in 800 μL of TNG buffer and 20μL of PMSI | ||
*Divide the preparation in 8 eppendorf (100μL) | *Divide the preparation in 8 eppendorf (100μL) | ||
| − | *For one eppendorf : add | + | *For one eppendorf : add glass bead, acid washed(150-212μm) |
*30 min on vortex | *30 min on vortex | ||
*Add 100μL of buffer in each tube | *Add 100μL of buffer in each tube | ||
| Line 42: | Line 42: | ||
| − | Read on a | + | Read on a plate at 488 nm excitation. |
{{Team:Paris_Saclay/notebook_footer}} | {{Team:Paris_Saclay/notebook_footer}} | ||
Revision as of 22:48, 19 October 2016
