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. Than run water on setting "high" on the cytometer.

• 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
• Assign FITC-positive cells to one histogram and FITC-negative to another
• Adjust Hoechst voltage to fit two-chromosome peak of FITC negative cells to channel 60 (make a vertical window with 60 in the middle and monitor the report)

=> The FITC positive cells show the peak than at about 40 (FITC quenches the Hoechst staining)

=> The numbers (volts, channel for - and + FITS) should be written in book

• Record xxxx events
• Change Threshhold to 2000 and the Hoechst voltage to move the two chromosome peak to 15 (This is for fast growing cells to not miss high numbers of chromosomes)

=> Write down parameters (FITC+ should be arround 10.5)

• Record xxxx events

Messure samples:

• Exchange standard with first sample
• Click on new sample???? and rename (short name since WinMDI has problems with long names)
• Watch the event number
• Move the peak of FITC - cells (standard) to 15
• Record xxxx events

Save data:

• Save data as fcs files at K:\Alle\KF_Flow\Montebello\Torsten Waldminghaus\
• Set the FITC data from log to linear
• Analyze data with WinMDI