Obtain calibration curve with fluorescent beads

 

Materials:

Methods:

1. Keep fluorescent beads refrigerated in C before use. Protect from light. Do not freeze.
2. Set up FACScan to the following parameters: ?????????
3. Vortex mix the vial contents to ensure that the polystryrene beads are uniformly suspended.
4. Add one drop from each vial to 2 mL of Haema-Line sheath fluid or buffered saline solution. Notice that vial components C and D from the low intensity kit are the same as vial components A and B from the normal intensity kit.
5. Sonicate the diluted bead suspension to disperse aggregates and vortex mix immediately before use.
6. Load unto FACScan, run for 10-60 seconds, then start recording data.
7. For each different fluorescent bead, should see a sharply defined peak. Experimental data:
8. Store bead fluorescense data for use in calibrating GFP fluorescense from live samples.