Posts filed under ‘Flow Cytometry’
Something we’ve found useful in analyzing our own data here at Cytobank is the ability to clone an experiment instead of having to download and re-upload files. If a colleague has shared an experiment with you and you don’t want to erase their hard work as you begin your analysis, make a clone! If you simply want to save time performing iterations of your own experiment analysis, make a clone! Experiment clones link back to the original experiments from which they were created on the Experiment Details page, so you’ll always have easy access to the original context. We’ve given you a variety of options for cloning, and you can find them under the “Cloning/Copying” section of the Actions box on the Experiment Details page.
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.
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.
Dataset #8414: Human Cord Blood – HSC isolation
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.
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…)
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.