September 22, 2011  |  Cytobank, Flow Cytometry

Deconvolute, Decode, Decipher! How to Split, Tag, and Analyze Your Barcoded Data on Cytobank

You may have heard about Fluorescent Cell Barcoding, a flow cytometry technique that allows researchers to answer a larger number of questions with the same amount of antibody, as compared to standard flow cytometry experiments [1,2]. We’ve prepared a few resources to help you learn about, perform, and analyze barcoding experiments.

Background

How does barcoding work? In the barcoding step, samples treated under different stimulation conditions are labeled with concentrations of dye that increase at a defined interval. The use of this dye to barcode effectively means that one cytometer channel is taken up for this code. The distinctly stimulated and labeled samples are then combined into one tube and stained with antibodies against targets of interest. This single tube is then run on a flow cytometer and data are collected for analysis. The most common approach is to barcode different stimulation conditions; however, barcoding can be applied to any distinct populations, such as patient samples or different time points of a stimulation condition.

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September 7, 2011  |  Cytobank, Flow Cytometry

Analyzing Hematopoietic Stem Cell Enrichment Data in Cytobank

Dataset #8414: Human Cord Blood – HSC isolation

Background

Hematopoietic Stem Cells (HSCs) give rise to all blood lineages and are capable of self-renewal. Clinically, HSC transplantation is under investigation for the treatment of diseases of the blood and bone marrow, including cancer, where a patient’s blood cells are wiped out and replaced with healthy cells that arise from transplanted donor HSCs. Transplant studies in mice have shown that only a few of these cells are necessary to repopulate the entire hematopoietic system.

Human umbilical cord blood is a rich source of stem cells, including HSCs. However, a variety of other cell types populate cord blood and must be removed from HSC preparations used for transplantation. Multipotent progenitor cells (MPPs) are one such population. Derived from HSCs, MPPs give rise to multiple lineages and are present in significant quantities in cord blood, though they are limited in their capacity for self-renewal. Purification of HSCs can be achieved by staining and running cord blood through a FACS sorter and isolating cells with a Lin-CD34+CD38-CD90+CD45RA- surface signature (as defined by Park, Majeti, and Weissman). MPPs can be quantified or isolated by their Lin-CD34+CD38-CD90-CD45RA- signature.

Sample Data

If you would like to try your hand at analyzing HSC enrichment data on Cytobank, we have made available an HSC dataset provided to us by scientists at BD Biosciences. You can find a tutorial to guide your analysis on our documentation site. More »

August 23, 2011  |  Cytobank, Education, Flow Cytometry

Future Proofing Your Experiments and Files: The Importance of Annotation

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:

  1. Tag your FCS files with key experiment details (e.g. “Patient-J01 IL-2 15m”)
  2. Label the channels you are measuring (before collecting data)
  3. Make sure scales and compensations work (before collecting data)
  4. Encode clinical sample IDs (don’t use HIPAA sensitive information)
Click the image to download as a PPT slide

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July 21, 2011  |  Cytobank, Flow Cytometry

Naming and Exporting Files in Diva

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!

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June 17, 2011  |  Announcements, Cytobank, Flow Cytometry

BD FACSelect™: Resources to Optimize Your Flow Experiments

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.

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June 12, 2011  |  Cytobank, Flow Cytometry

Never Email FCS Files Again!

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:

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June 10, 2011  |  Cytobank, Flow Cytometry

Working with Accuri Data

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.

Image plots from Accuri CFlow

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!

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May 20, 2011  |  Announcements, Flow Cytometry

What is SPADE?

What is SPADE?

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.

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