Lidstrom: Flow Cytometer
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Using the Flow Cytometer
- Turn on the Flow Cytometer System by flipping the bottom switch on the left side of the machine.
- Turn on Laser 1 (488 nm) by flipping the top switch on the left side of the machine. Laser 2 is controlled through software.
- Open FloMax Program on the computer (Ugly head Icon)
- Open Camera (CCD Camera)
- Flush System
- In the menu bar, go to “Acquisition,”
- Put waste container under the sample insertion tubes
- Select “Sheath Fluid Prime”
- Sample insertion tubes should drip
- Flush with 50 mL (~15 min)
- If cell sorting, flush sorting line out with 50 mL (~15 min) too
- Check Camera for bright spots (aka bubbles)
- Move tubing to try to dislodge bubbles
- If that fails, dethatch tubing and use syringe to remove bubble
- Flush system for 15 minutes after reattaching tubing
- Open Instrument Setting
- Go to Setup to set a number of cells to be counted by checking the enable button
- Hit Clean
- Push sample in cuvette up into chamber
- Push Clean
- Run bead standard (3 uM)
- Clean
- Calibrate with 1,3 and 5 uM beads separately (stored in fridge)
- 1 drop of bead solution/1-2 mL
- Repeat for all samples
- Reading the charts
- SSC v. FSC: shows size distribution of the particles
- SSC v. FL1: shows fluorescence separated by size from laser 1
- SSC v. FL2: shows fluorescence separated by size from laser 2
- If needing to break for more than 2 hours, put a cuvette of water in the sample chamber and press end
- After running samples, clean the machine by running the following through the machine
- 1 cuvette of wash solution
- 1 cuvette of 100% ethanol
- 1 cuvette with water
- Put new cuvette of water in the sample chamber
- Close the programs
- Turn off the Laser (top switch on left side)
- Turn off the System (bottom switch on left side)
- Shut down Flomax
Media Prep (Should be done the day before the run)
- Media without organic substrates should be used
- Filter media
- Autoclave media
- Attach to instrument while media is still hot
- Leave overnight
OR If not autoclaving, 1. Flush media with helium for 3-9 hours Make 20x stock à Appropriate amount of autoclaved water Mix while water is hot and attach to instrument