Spherotech COMPtrol Kit

Reagent preparation: 

Vortexing is required for proper resuspension of the individual kit components before use.

Procedure for bead staining: 

1. Add one drop of negative beads and one drop of positive beads to each test tube.

2. Place single color antibody conjugates into individual, labeled test tubes at the antibody concentration used for your application and vortex immediately.

3. Incubate at room temperature in the dark for 20 minutes.

4. Add 1 mL of buffer to each test tube and vortex. Centrifuge at 300 × G for 6 minutes. For best results, use the wash buffer described in the “Additional required equipment” section.

5. Decant the supernatant and resuspend the beads in 600 μL of wash buffer.

Compensation setup on Cytometer

1. Create an acquisition protocol with a Forward Scatter (Lin) vs. Side Scatter (Lin) dot plot and either single color histograms or dual color dot plots for each relevant fluorescence channel. On the Forward

Scatter vs. Side Scatter dot plot, create a region to capture the bead population and gate all fluorescence plots/histograms on that bead population. Set the protocol to collect 10,000 beads. Forward Scatter and Side Scatter adjustment may be needed in order to see the beads on the plot.

[NOTE]

Verify that the discriminator (or threshold) is low enough to detect the bead population.

2. Using cytometer settings optimized for your application, run each of the stained bead samples and ensure that the positive and negative signals are on scale.

3. Generate a compensation matrix using acquisition and/or analysis software.

[NOTE]

If large amounts of doublets and triplets are visible in the Forward Scatter vs Side Scatter dot plots, sonicate the stained sample for 15-20 seconds and reacquire.