User:Torsten Waldminghaus/flow cytometry notes

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).

1xTBS

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:

1. Count/SSCA
2. Count/Hoechst
3. Count/FITC
4. Count/Hoechst

• Go to Instruments > Parameters > Voltage:
  • SSC: 500
  • Hoechst: adjust later using the standart /linear scale
  • FITC: 580 /log scale
• Go to Instruments > Threshhold:
  • Press Add two times; delete SSCA and change Hoechst to 5000 for Standard (for samples it should be 2000)
• Go to laser and set windows ext. to 2000
• UV delay is 40.000 and can be changed if one gets strange results.

Recording the standard:

• Press "low" at cytometer and insert standard cells
• Number of events (number of cells counted) should be adjusted with the smal weel to about 1000-1500 (per second)
• The dot plot shows two populations: one with FITC staining and one without
• Lay two windows (vertical) over the dot plot to seperate the two populations