Table 2. Troubleshooting table.

Step	Problem	Possible reason	Solution
3, 14, 15, 17	Fluorochrome ‘spillover’ (i.e. non-specific fluorescence detected in a neighboring channel, e.g. false-positive events in the PE channel attributable to FITC fluorescence)	Improper selection of fluorochrome, or inadequate understanding of antigen and epitope distribution. Improper instrument setup and fluorochrome compensation. Use of tandem dyes (e.g. PE-Cy5).	Check fluorescence spectrum viewer (http://bdbiosciences.com/spectra or http://www.invitrogen.com/site/us/en/home/support/Research-Tools/Fluorescence-SpectraViewer.html). Examine single- color staining profiles and perform ‘fluorescence minus one’ or FMO36 control stainings to identify the culprit fluorochrome. Knowing the distribution of antigens and epitopes targeted by antibodies, and having a proper understanding of fluorochromes, their selection and instrument setup and operation are crucial for polychromatic flow cytometry10,34-39. Refer also to instrument user’s guide provided by the flow cytometer manufacturer.
3B, 17	Dull staining of manually conjugated Pacific Blue antibody (e.g. anti-CD123) using the Invitrogen Zenon labeling kit	i. Suboptimal antibody: conjugate ratio.	Optimize amount of Component A used, and titrate antibody levels. Component B (blocking immunoglobulin) must also be adjusted stoichiometrically (refer to manufacturer’s instructions). Alternatively, a Pacific Blue equivalent such as eFluor 450 can be used.
7, 11, 17	Lack of consistency in phenotype readout.	Donor variation or instability of phenotype. Lack of a consistent time interval between cell fixation and flow cytometric acquisition. Use of non-standardized, non-fresh reagents.	Allow samples to stabilize in fixative for at least 1 h after immunostaining. Analyze at a fixed time interval (e.g. 16 h after cell fixation). Avoid analysing later than 24 h as fluorescence intensity or tandem dyes might begin to degrade. Use freshly prepared standardized reagents (e.g. BD FACS lysing solution, CellFIX).
7, 17	Odd or variable FSC vs SSC bivariate profiles or large amounts of debris, including dead cells.	Prolonged incubation (>15 min) with FACS Lysing Solution can alter cellular biophysical properties or surface epitopes.	Stop incubation promptly at 10 min, and invert tubes less vigorously.
9, 11, 17	Low cell yield.	Discarding supernatant without care. Rarity of cell population.	Pipet off supernatant carefully if using microcentrifuge tubes, or pour away supernatant gently when using FACS tubes. Consider scaling up blood volumes and staining reagents.
14, 17	Progressive shifting of acquisition events on FSC vs SSC bivariate plot over days and sometimes during a data collection session. This can	Obstruction of internal tubings that supply or drain flow cell. Fluidics or pressure problems in tubing circuit.	Ferromagnetic valves are often used to regulate pressures and buffer or waste flow within certain flow cytometers. Use of magnetic bead- based cell sorting and subsequent analysis on such flow cytometers can cause valves to malfunction, leading to increased internal pressure within the flow cell. Discontinue the use of