Tips: Data Analysis

Cell aggregates will take longer to pass through the laser intercept than single cells. This affects the width and the area of the signal pulse, while the height stays roughly the same. Using a pulse geometry gate (such as FSC-A vs. FSC-H or FSC-W vs. FSC-H), doublets and larger cell aggregates can be eliminated.
Nuclear dyes (such as DAPI, Sytox dyes etc.) can be used for such a pulse geometry gate as well (for example as DAPI-H vs. DAPI-W), particularly convenient for populations with heterogenous size/complexity, as well as small particles (as SSC and FSC signals are less sensitive in that range). 

In sorting, it is particularly important that you include a doublet discrimination gate (two are preferable), as these mistakes cannot be indone and sorted doublets might decrease the purity of your sorted populations. 

If the area and height intensities of an event do not match (and an -A vs -H plot looks skewed), this means the Area Scaling Factor is not set correctly for the type of cell. This will have a negative impact on sorting quality. Please contact us if you need help setting this up. 

For reliable data, an initial quality control step should be performed on the flow cytometry data: looking at the data information versus time. This reveals fluidics and signal instabilites resulting from air bubbles or pressure changes. A time vs. scatter plot visualizes the data over time and allows to eliminate artefacts. 

In fact, also FlowJo has implemented this procedure, called 'Check Sample Quality' or 'FlowClean' plugin in the software. 

There are many different software sources on the market that can be used for post-acquisition analysis of flow cytometry data, in additon to using the software on the cytometer PC.

available via the CFFlowCyt:

• SpectroFlo * - data analysis software of Cytek Aurora
• Flowing Software * (Perttu Terho, Cell Imaging Core of the Turku Centre for Biotechnology - free for anyone) 
• FlowJo (Tree Star, Inc.) in the context of data analysis service by the facility staff

others:

• FCSExpress (DeNovo Software)
• ModFit (Verity Software House) - specialized on cell cycle analysis and cell tracking
• Cytobank (Cytobank Inc.) - Premium and Enterprise version with different functionalities 
• Astrolabe - high dimensional data analysis service

The 'DailyDongle' Blog by FlowJo provides news about the software itself, but also features free webinars and general analysis and troubleshooting tips. 

For data generated on the Amnis ImageStream, the corresponding IDEAS® software can be used. Data files for imging cytometry are large and therefore demand enough computing power. The Core Facility provides a computer workstation equipped with the IDEAS® software specifically for the analysis of imaging cytometry data. The computer can be booked via the PPMS booking system. 

Decreasing the file sizes by deselecting all unnecessary parameters during acquisition safes computing time.

As with data sets from other techniques (transcriptomics, RNAseq...), conducting analysis on already collected data, re-examining it and expanding the readouts leads to invaluable synergy in the research environment. The prerequisite is a (semi-)public tool for storage, analysis and data representation. 

FlowRepository.org was developed as a public flow cytometry data repository in order to enable access to published data including associated metadata and facilitate its review. Spidlen et al. specify: 'Through FlowRepository, users can access, review, download, deposit, annotate, share, and analyze flow cytometry datasets. Since proper annotation is key to third-party interpretation, FlowRepository supports all annotation concepts specified by the Minimum Information about a Flow Cytometry Experiment (MIFlowCyt)'.

For researchers, this now allows access to an abundance of data from already performed flow cytometric experiments in order to re-analyze and verify/complement their approaches and findings.
In fact, publishing flow data like this is now mandatory for publication in Open Access journals such as PLOS ONE.

Important information about the FlowRepository include

• Spidlen et al., Preparing a Minimum Information about a Flow Cytometry Experiment (MIFlowCyt) Compliant Manuscript Using the International Society for Advancement of Cytometry (ISAC) FCS File Repository (FlowRepository.org). Current Protocols in Cytometry 2012
• Spidlen and Brinkman. Use FlowRepository to share your clinical data upon study publication. Cytometry B Clin Cytom 2016 
  

'A fundamental tenet of scientific research is that published results are open to independent validation and refutation. Minimum data standards aid data providers, users, and publishers by providing a specification of what is required to unambiguously interpret experimental findings.' (excerpt from Lee at al., MIFlowCyt: The Minimum Information About a Flow Cytometry Experiment. Cytometry Part A)

The standards for publishing flow cytometry data were compiled by experts in the field and vetted by the International Society for the Advancement of Cytometry (ISAC). The so-called MIFlowCyt standard recommends descriptions of specimens, controls, reagents, system configuration, instrumental quality control experiments and data processing. Using the standard is required/recommended by the journals Cytometry A and Nature, amongst others.
Regarding publishing flow cytometry data, Nature specifies rules for data presentation, for example that all plots have to be 'contour plots with outliers or pseudocolor plots'. 

Publications describing the standards include:

Lee et al., MIFlowCyt: The Minimum Information About a Flow Cytometry Experiment. Cytometry Part A 2008

Spidlen et al., Flow Cytometry Standards. BMC Res Notes 2011

Remember to acknowledge the Core Facility!