Frequently asked questions (FAQ)

To reduce cell clumping, always filter your samples prior to sorting. Clumps and aggregates can lead to reduced event counts and poor staining quality due to doublet discrimination. It is strongly recommended to include a viability dye to exclude dead cells from analysis.

For the sample buffer, we suggest using PBS supplemented with 1% FBS or 0.5% BSA, along with 1 mM EDTA to minimize aggregation. A standard FACS buffer is generally recommended. Avoid using higher concentrations of serum or BSA, as this may interfere with optical resolution.

Successful panel design starts with understanding your biological question and the relative expression levels of your target markers. Bright fluorochromes (e.g. PE, APC) should be assigned to low-abundance markers, while dimmer ones (e.g. FITC, Pacific Blue) can be used for highly expressed antigens.

Minimize spectral overlap by avoiding fluorochromes with similar emission spectra on co-expressed markers. Use proper controls (single-stained, FMO, viability) and check the laser configuration and the respective filters for the available cytometers beforehand to ensure compatibility.

If you are unsure about fluorochrome combinations or panel complexity, we recommend reaching out to the Flow Unit for guidance.

Please bring your cell suspension in an appropriate sample buffer (e.g. FACS buffer). If you are planning to sort, make sure to bring collection tubes containing the desired collection medium or buffer, as well as an ice box with a lid to protect your samples from light and maintain temperature while waiting.

Additional sample buffer is recommended in case the sample needs to be diluted. If you are running a staining panel, bring your panel design and gating strategy. For compensation, don’t forget your single-stained controls and an appropriate unstained control.

Keep in mind: you are the expert on your experiment - we are here to support you in putting your ideas into practice.

In most cases, we recommend using compensation beads for single-stain controls. Two commonly used options include:

• UltraComp eBeads™ (Thermo Fisher)
• BD CompBeads (BD Biosciences)

These beads are antibody-capture particles that provide consistent, bright signals and are suitable for most fluorochrome-conjugated antibodies. However, in some cases, especially when working with fluorescent proteins like GFP, YFP, or tdTomato, cell-based controls may be necessary. If you are using cells for compensation, please also provide an unstained cell control.

General Guidelines:

• You will need one single-stain control for each fluorochrome in your panel, plus one unstained control (usually unstained beads or cells, depending on what you are using).
• Compensation controls must be as bright or brighter than the corresponding signal in your experimental samples. Low-abundance antigens (e.g., activation markers, cytokines) may not be suitable for compensation.
• For tandem dyes (e.g., PE-Cy7, APC-Cy7), the antibody clone and fluorochrome used for the real staining and compensation must be identical, as tandem dyes are sensitive to lot-to-lot variation and may degrade over time.
• Compensation controls must include a sufficient percentage of positive events. This can be a challenge with cell-based controls; in such cases, researchers sometimes substitute with another antibody conjugated to the same fluorochrome (e.g., CD45-PE instead of CD34-PE).
  → Important: This is only acceptable if the substituted antigen is expressed at higher levels than the original and should never be used with tandem dyes.

If you are unsure about your setup, feel free to contact the Flow Unit before your appointment.

Clogging: Caused by cell aggregates or unfiltered samples. Always filter through a 35–70 µm mesh and use EDTA containing buffer to reduce clumping. If required, please contact our responsible TAs. 

Low or no event rate: May result from low cell concentration, clogs, or air in the system. Check sample quality and system fluidics.

Unstable stream (sorting): Often due to nozzle clogs or air bubbles. Clean the nozzle and make sure sample tubes are filled properly.

Weak or no signal: Check antibody quality, staining protocol, and ensure lasers and filters match your fluorochromes.

Compensation issues: Use bright, proper single-stain controls; ensure fluorochrome identity matches exactly, especially for tandem dyes.

High background: Caused by dead cells or nonspecific binding,  include viability dyes and consider blocking steps.

If you are interested in using one of our instruments, please reach out to us at flowcytometry(at)dzne.de.
Once you have received training from a member of the Flow Unit, you will be granted access to the internal booking system and can schedule your sessions as usual.

That depends on your needs. Training sessions are time-intensive and typically span several hours. If you only require access once or twice a year, it may be more practical for the Flow Unit to assist with acquisition or sorting. However, if you plan to use the instrument regularly, we are happy to provide training so you can work independently.