Lidstrom: Flow Cytometer

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Using the Flow Cytometer 1. Turn on the Flow Cytometer System by flipping the bottom switch on the left side of the machine. 2. Turn on Laser 1 (488 nm) by flipping the top switch on the left side of the machine. Laser 2 is controlled through software. 3. Open FloMax Program on the computer (Ugly head Icon) 4. Open Camera (CCD Camera) 5. Flush System a. In the menu bar, go to “Acquisition,” b. Put waste container under the sample insertion tubes c. Select “Sheath Fluid Prime” d. Sample insertion tubes should drip e. Flush with 50 mL (~15 min) f. If cell sorting, flush sorting line out with 50 mL (~15 min) too 6. Check Camera for bright spots (aka bubbles) a. Move tubing to try to dislodge bubbles b. If that fails, dethatch tubing and use syringe to remove bubble c. Flush system for 15 minutes after reattaching tubing 7. Open Instrument Setting 8. Go to Setup to set a number of cells to be counted by checking the enable button   9. Hit Clean 10. Push sample in cuvette up into chamber 11. Push Clean 12. Run bead standard (3 uM) 13. Clean 14. Calibrate with 1,3 and 5 uM beads separately (stored in fridge) a. 1 drop of bead solution/1-2 mL 15. Repeat for all samples 16. Reading the charts a. SSC v. FSC: shows size distribution of the particles b. SSC v. FL1: shows fluorescence separated by size from laser 1 c. SSC v. FL2: shows fluorescence separated by size from laser 2 17. If needing to break for more than 2 hours, put a cuvette of water in the sample chamber and press end 18. After running samples, clean the machine by running the following through the machine a. 1 cuvette of wash solution b. 1 cuvette of 100% ethanol c. 1 cuvette with water 19. Put new cuvette of water in the sample chamber 20. Close the programs 21. Turn off the Laser (top switch on left side) 22. Turn off the System (bottom switch on left side) 23. Shut down Flomax   Media Prep (Should be done the day before the run) Media without organic substrates should be used 1. Filter media 2. Autoclave media 3. Attach to instrument while media is still hot 4. 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

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