User:Torsten Waldminghaus/flow cytometry notes: Difference between revisions
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==Recipe== | ==Recipe for flow-TBS== | ||
*1x TBS with 20mM Tris used for flow cytometry in Skarstad-Lab | *1x TBS with 20mM Tris used for flow cytometry in Skarstad-Lab (reduced NaCl concentration from 150mM in the usual [[TBS]] to 130mM (everything else is the same). | ||
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*Prepare and sterile filter. Store at 4°C. | *Prepare and sterile filter. Store at 4°C. | ||
==Use of flow cytometer BD LSR II== | |||
*When started new no washing is required but before starting with samples one should press ''''Prime' without any liquid''' (never have 'Prime' with liquid!!!' to let run air run through the system. | |||
*Start software FACSDiva | |||
*Select Instrument Configuration | |||
**Select presettings of Gunnar F | |||
*Go to Cellbiology > Torsten > new exp. (brown icon) > rename (torsten date)> | |||
*press syringe icon | |||
*go to 'tube_001' > things will show up on right screen | |||
*make local sheet by pressing on icon on left top | |||
*Create diagrams: | |||
**Click on DotPlot icon and than on the sheet | |||
**Axes can be changed by clicking on the axe-title | |||
**Make dot plot with FITC versus Hoechst | |||
**Make 4 histograms: | |||
#Count/SSCA | |||
#Cont/Hoechst | |||
#Cont/FITC | |||
#Cont/Hoechst | |||
*Go | |||
Revision as of 05:20, 14 April 2008
Recipe for flow-TBS
- 1x TBS with 20mM Tris used for flow cytometry in Skarstad-Lab (reduced NaCl concentration from 150mM in the usual TBS to 130mM (everything else is the same).
| for 500 ml |
|---|
| 10 ml 1M Tris-HCl pH 7.5 |
| 13 ml 5M NaCl |
| 477 ml ddH2O |
- Prepare and sterile filter. Store at 4°C.
Use of flow cytometer BD LSR II
- When started new no washing is required but before starting with samples one should press 'Prime' without any liquid (never have 'Prime' with liquid!!!' to let run air run through the system.
- Start software FACSDiva
- Select Instrument Configuration
- Select presettings of Gunnar F
- Go to Cellbiology > Torsten > new exp. (brown icon) > rename (torsten date)>
- press syringe icon
- go to 'tube_001' > things will show up on right screen
- make local sheet by pressing on icon on left top
- Create diagrams:
- Click on DotPlot icon and than on the sheet
- Axes can be changed by clicking on the axe-title
- Make dot plot with FITC versus Hoechst
- Make 4 histograms:
- Count/SSCA
- Cont/Hoechst
- Cont/FITC
- Cont/Hoechst
- Go
