How much Ab should be used to stain Quantum Simply Cellular (QSC) beads
Posted: January 30, 2017
It’s advisable to either add a sure excess of antibody, or conduct a titration to ensure saturation. In looking over historical data for QSC anti-Mouse labeled with conjugated primary antibodies, concentrations vary quite a bit (depending on clone and fluorochrome). In one titration study, per-population antibody concentrations varied from 0.032µg Ab / population - 1.25µg Ab / population. Factors to consider include the titration curve, appearance of the histograms, and details of the antibody conjugate(s) (e.g. concentration, specific flourophores [MW, etc.]). PE has such a large MW (240kD) that it makes a significant contribution to the mass of the conjugate. Our datasheet on Antibody Titration provides a general protocol, and our Tips & Troubleshooting piece provides additional guidance.
You can use the highest Ab concentration to saturate all bead populations, though you should look at the quality of the histograms to ensure that peaks aren’t becoming broad due to nonspecific binding.
We recommend the same fluorescence settings for beads and cells, i.e. PMT voltages and compensation settings. Gating will be different between beads and cells, and FSC/SSC settings may be different as these do not impact fluorescence measurements.