Quick-start configuration

Find your cytometer model in the table below. The four columns tell you exactly what to use for forward scatter, bead classification, reporter detection, and whether color compensation is required.

Cytometer not listed? The platform is compatible with any flow cytometer that meets the minimum requirements described in the next section. Contact our technical team with your model number and we'll confirm channel assignments and provide configuration support.

Channel selection logic

If your cytometer isn't in the quick-start table, the principles below let you map the assay onto any compatible instrument.

Bead classification channel

GeniePlex capture beads carry a far-red fluorescent dye with peak emission near 700 nm. Detect it on whichever of these channels your instrument offers, in order of preference:

• PE-Cy5 channel with blue (488 nm) excitation — most common configuration, works on the majority of cytometers.
• APC channel with red (633 or 640 nm) excitation — preferred when available; gives cleaner separation from PE.
• PE-Cy7 channel with blue excitation — acceptable alternative when neither of the above is available.

PE reporter channel

The reporter dye is phycoerythrin (PE), with peak emission at ~575 nm and excitation by the blue (488 nm) laser. Use whichever channel your instrument designates as "PE", "FL2", or "Yellow" depending on the manufacturer's labelling convention.

Setup bead preparation

Before running samples, you'll prepare two setup tubes: blank beads for initial gating and PMT voltage tuning, and Standard 1 beads for verifying signal on the PE axis and applying compensation.

Blank beads

Aliquot half of the resuspended bead suspension from a Blank well (typically ~75 µL if the assay resuspension volume was 150 µL). Transfer to a sample tube or 96-well plate well, depending on your cytometer's sample loading mechanism. Add 100–300 µL of 1× Reading Buffer.

Remaining capture bead working suspension from the assay run can be substituted: 45 µL of the working suspension plus 100–500 µL of 1× Reading Buffer.

Standard 1 beads

Aliquot half of the resuspended bead suspension from a Standard 1 well (~75 µL). Add 100–300 µL of 1× Reading Buffer to the tube or plate well.

Volume note: If you add Reading Buffer to the Blank and Standard 1 wells directly (75–150 µL), bead concentration during acquisition will be lower and the run slightly slower — this is acceptable but plan your acquisition rate accordingly.

Display layout & voltage setup

Begin with instrument startup, fluidic stability verification, and optical alignment per your cytometer manufacturer's recommendations. Run calibration particles (e.g. Spherotech Rainbow Calibration Particles) before proceeding.

Create the protocol layout

Open a new acquisition protocol and create the following plots:

1. One dot plot: FS (X-axis) × SS (Y-axis), linear display.
2. Two histograms on the bead classification channel (PE-Cy5 or APC), log display.
3. Two dot plots: PE (X-axis) × bead classification channel (Y-axis), log display.
4. If both PE-Cy5 and APC (or PE-Cy7) channels are available, duplicate the histograms and PE dot plots for the second channel as well — useful for direct comparison.

Set all compensation values to zero at this stage. Enable acquisition of both Area and Height for all fluorescence parameters. Save the protocol.

Tune PMT voltages with blank beads

1. Adjust FS and SS gains

   Run the prepared blank bead sample. Adjust forward and side scatter gains until both bead size populations are on-scale on the FS × SS plot.

2. Create gates for the two bead sizes

   Draw Gate 1 around the smaller 4-micron beads (S4) and Gate 2 around the larger 5-micron beads (S5). Apply Gate 1 to one bead classification histogram and one PE × classification dot plot. Apply Gate 2 to the other set.

3. Adjust bead classification PMT voltage

   Tune the PMT voltage on the bead classification channel until all peak populations within each gate are clearly resolved on the histograms and dot plots. For example, a typical S4 gate resolves four bead populations labelled S4P3, S4P7, S4P9, and S4P11, where P3 is the dimmest and P11 the brightest peak.

4. Adjust PE PMT voltage

   Tune the PE channel voltage so the dimmest bead population sits within the first decade on the PE-axis. This ensures unstimulated samples generate measurable but unsaturated signal across the dynamic range.

5. Save the protocol

   Saving at this point captures your tuned baseline before applying compensation in the next step.

Verify with Standard 1 beads

Run the prepared Standard 1 setup sample using the protocol saved in the previous section.

1. Confirm all bead populations on the PE-axis are on-scale — none should be saturated at the upper limit of detection.
2. If any population is off-scale, reduce the PE PMT voltage incrementally. After each adjustment, re-verify that the dimmest population is still visible above noise on the PE × classification dot plot.
3. If you've adjusted PE voltage, plan to re-run Standard 8 (highest concentration standard) before acquiring samples, to confirm the top of the standard curve is also within range.

Color compensation

Color compensation is required only on instruments where the bead classification dye (peak ~700 nm) spills into the PE channel (~575 nm). The quick-start table in Section 01 indicates which instruments require it.

Applying compensation

With Standard 1 beads running, adjust the % PE − classification channel compensation value until the bead populations sit in a horizontal orientation on the PE × classification dot plot — meaning bead position along the PE axis is independent of bead classification intensity.

Multi-channel setup

If you're configuring both PE-Cy5 and a secondary channel (PE-Cy7 or APC) for cross-validation or comparison, repeat the voltage tuning and compensation steps for each channel independently. Compensation between PE and APC is typically zero or minimal because the spectral separation is large.

Save the final protocol once compensation is set. Your instrument is now configured to acquire samples.

Validated configurations

The following table summarises cytometer/sample/analyte combinations published by GeniePlex users. Use it to confirm your intended configuration has direct precedent — or as a reference when designing a similar study. Gold-highlighted rows are from featured citations that explicitly cite GeniePlex in their methods.

Human samples

Mouse samples

Rat samples

Troubleshooting

Common setup issues and how to resolve them. If your problem isn't listed here, contact technical support with your instrument model, panel SKU, and a screenshot of the affected plot.