TITLE: Instructions for CytoFLEX “Ready to Use” (RTU) QC Calibration Fluorospheres
AUTHORS: Jamie C Tijerina

[DESCRIPTION]
The CytoFLEX "Ready To Use" (RTU) QC Calibration Fluorospheres do not need to be diluted which reduces QC failures that are related to bead preparation errors. This protocol has been tested in the Flow Cytometry and Cell Sorting Facility at Caltech to ensure optimal results during QC in both plate and tube mode, while minimizing waste through reduction of the amount of beads that are discarded following QC, and minimizing plastic waste. Testing determined that one drop of beads deposited from the bottle is sufficient to run one test, however there must be enough placed in the tube or well to run a QC. In plate mode, the suggested minimum sample volume per well is 45ul. In tube mode, the suggested minimum sample volume is 100ul. These beads should be stored in a standard refrigerator close to the instrument. This protocol and workflow can be used in a core facility, or by an individual lab that has its own CytoFLEX instrument. 

[GUIDELINES]
Beads should be stored in a standard refrigerator near the instrument. 
To reduce plastic waste, a 96-well flat bottom plate can be kept near the instrument to be used only for the purpose of running QC and cleaning procedures.

[STEPS]
SECTION: To Run QC in Plate Mode
1. Remove the RTU QC Calibration fluorospheres box from the refrigerator.
SECTION: To Run QC in Plate Mode
2. Check the bead lot number on the bottle or on the side of the box. Under the drop-down menu for the “bead lot,” ensure that the bead lot on the box matches your selection.
SECTION: To Run QC in Plate Mode
3. In the A1 well of a 96-well flat bottom plate, deposit 2 drops of RTU QC Calibration Fluorospheres directly from the bottle.
SECTION: To Run QC in Plate Mode
4. Insert the plate into the loader on the instrument.
SECTION: To Run QC in Plate Mode
5. Run QC.
SECTION: To Run QC in Plate Mode
6. Return the box with the fluorospheres to the refrigerator.
SECTION: To Run QC in Tube Mode
7. Remove RTU QC Calibration fluorospheres from the refrigerator.
SECTION: To Run QC in Tube Mode
8. An aliquot of beads will be measured out by Facility staff and placed in a 5mL tube at the center of the box of beads. Please use the aliquot to run your QC. The purpose of this is to reduce waste in light of persistent global supply chain issues. The aliquot is prepared to the standard set by Beckman Coulter, which is 10 drops.
SECTION: To Run QC in Tube Mode
9. Check the bead lot number on the aliquot tube. Under the drop-down menu for the “bead lot,” ensure that the bead lot on the tube matches your selection.
SECTION: To Run QC in Tube Mode
10. Remove the cap and place the tube on the sample loader.
SECTION: To Run QC in Tube Mode
11. Run QC.
SECTION: To Run QC in Tube Mode
12. Place the aliquot back in the box, and return the box to the refrigerator.
SECTION: To Run QC in Tube Mode
8.1. If the aliquot has been damaged or runs out, please prepare a fresh tube by squeezing 10 drops from the bottle directly into a 12x75 polypropylene or polystyrene tube. Do not discard after the run. Please label it with the date it was prepared, lot number, and your initials using lab tape and a marker. The tube may be stored in the refrigerator closed with a cap.