Ever find yourself staring at a folder of FCS files and thinking, “Wait, now which tubes did I add PMA to, how much did I add, and which samples were these again?”
Jonathan from Cytobank/Stanford recommends what he calls “future proofing” in order to avoid this problem. He explained this approach during a CYTO 2011 Pre-Congress course in his talk titled “Flood Cytometry: Embracing Single Cell Systems Biology (and coping with large cytometry experiments).” In that talk, he outlined four easy steps that are useful for experiments of all sizes.
When collecting on the cytometer:
Tag your FCS files with key experiment details (e.g. “Patient-J01 IL-2 15m”)
Label the channels you are measuring (before collecting data)
Make sure scales and compensations work (before collecting data)
Encode clinical sample IDs (don’t use HIPAA sensitive information)
Many of our users upload data to Cytobank that were collected on a BD Biosciences flow cytometer using BD FACSDiva™ software. In this post, we will walk you through how to harness the power of naming files in Diva to facilitate analysis on Cytobank and how to export FCS files from Diva for upload to Cytobank.
Detailed naming of sample files in Diva gives you a special advantage unique to Cytobank’s analysis environment. When you upload files to Cytobank, our servers will automatically categorize your files for ease of analysis when you annotate Figure Dimensions. This automated categorization enables you to rapidly generate well-annotated plot layouts and figures. Watch our YouTube video demonstrating this feature!
Designing a successful flow experiment – selecting compatible reagents and optimizing your protocol – can be challenging and time-consuming. And yet, as we all know, a well-designed experiment is critical to the collection of high-quality flow data.
What do we think about when designing flow experiments?
What buffers should I use when probing intracellular targets?
Which surface antibodies work well on my sample and with my buffers?
What is the best concentration for my antibody?
Are there alternative protocols that work better with my samples?
We are excited to announce the arrival of two resources that will help you answer those questions and streamline your reagent selection process. BD Biosciences has released the FACSelect™ series, consisting of a Multicolor Panel Designer and a Buffer Compatibility Resource.
Are you working with a collaborator who needs to see your raw data? Are you looking for help from a Cytobank administrator relating to experiment analysis?
Don’t bother opening your email client, searching for an email address, and digging through folders for your flow files. Instead, use the easy sharing features built into Cytobank. Once you have uploaded files to your account, they can be easily shared with others from within the Cytobank interface.
As always, your experiment is visible only to you until you actively choose to give permission to another user to see it. When you do choose to share an experiment, follow these easy steps:
Cytobank users have uploaded and analyzed data collected from more than 30 different flow cytometer models, so chances are that Cytobank can handle your data! In a recent post, we featured the ability of Cytobank to facilitate the mining of data from large datasets generated by the DVS Sciences CyTOF. This time, we will walk you through analysis of data collected on the Accuri cytometers using their CFlow software.
Accuri provided us with a set of sample files demonstrating the collection of data from cells stained with a PE-anti-CD4 antibody, and we’ll use this as an example. You can see from their CFlow software analysis that they achieve separation of and gate on the lymphocyte population (P1, first panel), and further separate CD4+ from CD4- cells (second two panels). We’ll show you how to do the same in Cytobank!
SPADE (Spanning-tree Progression Analysis of Density-normalized Events) is a way to automatically identify populations in multidimensional flow cytometry data files. SPADE clusters cells into populations and then projects them into a tree like the one shown below. SPADE works for data from both ‘classic’ fluorescence flow cytometry and mass cytometry.
Mass cytometry, a technique developed by DVS Sciences, now a Fluidigm Company represents a revolutionary spin on classic fluorescence-based flow cytometry. Instead of using antibodies tagged with fluorophores (in which spectral overlap quickly limits the number of parameters available for simultaneous detection), mass cytometry relies on antibodies tagged with transition element isotopes. Antibody-bound cells are vaporized, ionized, and analyzed on a mass spectrometer.