Honeychrome: supported cytometers

First off, if you've used Honeychrome, we'd appreciate feedback. I've tried to create this feedback form, which hopefully works as intended. If you've tried it with your data, or the example data, did it work? Did you run into issues? What did you like or dislike about the experience of working with the software?

Users have reported that testing on FCS files from different instruments have revealed issues due to lack of conformity, but never fear... we are immediately rolling out a feature to clean up these issues.

We've now tested and support these spectral cytometers:

1. Cytek Aurora & Northern Lights

2. Sony ID7000

3. BD FACSDiscover A8 & S8

4. BD FACSymphony A5SE

5. Beckman Coulter CytoFLEX mosaic

6. ThermoFisher Attune Xenith

7. NovoCyte Opteon

The exported FCS files should now be fully FCS 3.1 compliant, and will include all the relevant metadata such as marker labels and fluorophore names. You'll also get the spectral profiles and any autofluorescence profiles written to the metadata of the FCS files, since we view these as essential to unmixing reproducibility. Imaging parameters from the FACSDiscover A8 and S8 data should be retained as-is in the unmixed data files.

Okay, so what do you need to do? You need to uninstall your current Honeychrome, and download and install the update. Don't worry, this doesn't delete your experiments. Your experiments are all managed by ".kit" files in an "Experiments" folder at the base of your user directory on your computer. If you install a new version of Honeychrome, it finds everything just as you left it. That said, if you had errors with an experiment that were causing it to become unresponsive or crash the app, those may persist, so for anything like that, I'd recommend starting over.

Steps:

1) Download and install again.

2) Open Honeychrome, open an experiment.

3) Recommended: refresh the experiment. This will reset gating and unmixing, but if you are working with something other than the Aurora or ID7000, this is probably necessary. Select File: Import FCS files. Then click "Update Experiment Configuration" in the lower right corner.

Right, what has changed?

1. The default scatter parameters for gating cells and singlets have been specified for each cytometer. You can change these to other channels, if you wish.

2. Default zoom view has been set for scatter on some instruments. For new instruments like the BD FACSDiscover and Novocyte Opteon, there is a huge working range on the scatter channels, which can be a pain to navigate. I've tried to set reasonable default views for working with leukocytes. These don't cap the data or restrict you from changing the scales, they just try to make it so you are close to the right area to start with.

3. Explicit definition of which channels are used for spectral unmixing.

For the scatter plots, there is a neat feature in Honeychrome to assist you with getting the data scaled on the plot nicely. Right-click on the plot and select "Fit Axes to Data".

This scales the data so it's onscreen, but keeps the gate in effectively the same place on the viewer. Very handy for getting started.

After fitting the axes to the data

Here are some spectral ribbon plots from the various spectral cytometers in the latest Honeychrome:

Sony ID7000 (6-laser data)

Note: for the ID7000, only every other detector is labeled on the plot because there are so many detectors. All of the detectors are plotted and used.

BD FACSDiscover A8

BD FACSymphony A5SE

Cytek Aurora

Cytek Northern Lights

NovoCyte Opteon

ThermoFisher Attune Xenith

Beckman Coulter CytoFLEX mosaic

Note: The CytoFLEX mosaic data seem to be on a smaller scale than I'm used to from R. I haven't worked with this instrument enough to know what the normal range is. If you're using this and the scale seems off, get in touch at the GitHub Issues page.

If you're working with one of these instruments, have installed the update, updated the experiment configuration and it still shows the wrong detectors, try deleting and creating the experiment anew. If that doesn't fix it, add it to the Issue page.

Currently, when exporting on MacOS, the folders may not show up correctly. If you have set up your experiment by linking it to existing data folders, as shown in the last post, these folders will appear, but they will be empty. Selecting these for export will not export any FCS files. Instead, select the option starting with "Link_", which links to your actual data. We will try to fix this for the next update.