#Compensation matrix flowjo how to
#Compensation matrix flowjo free

There are a lot of ways to get bad flow cytometry data – in this post I’ll focus on the reasons that I feel are the easiest to identify and fix. So in this post I’d like to go over some examples of bad data to assist novice flow cytometrists. It was just a skill that I eventually picked up. Looking back, I realized that no one really ever told me what bad data looks like. But for novice flow cytometrists this step of identifying problems can be very difficult. With baking this is easy for everyone, even a novice – you taste your food and immediately know it doesn’t taste good.

#Compensation matrix flowjo how to

The step that is often skipped is how to assess your results to identify problems. Flow cytometry training materials usually focus on the protocol of how to perform a flow cytometry experiment, but they don’t always cover how to troubleshoot. As I created my flow cytometry training materials I realized that I didn’t cover all of these steps. It’s been a while since I learned flow cytometry, but baking bread reminded me of the process of learning a new skill: follow a protocol, assess the results and identify problems, determine how to fix the problems, and repeat the protocol with adjustments to address the problems.

#Compensation matrix flowjo free

And while I learned to perfect my bread in my free time, I spent a lot of my working hours on training materials for new flow cytometrists – that’s when I started to realize some similarities between these two tasks. I decided to take on baking sourdough bread (like many other quarantined individuals). When you modify the matrix, the numbers are converted to give an estimation of the amount (percentage) of spillover into the other parameter.When the global pandemic hit and Chicago’s shelter in place orders prevented me from going into the lab, I started doing some experiments in my kitchen instead. In version 4.5 and after, the matrix can be modified right in FlowJo using the 'Edit/Save Matrix' menu option. In very rough terms, the 0.1803 corresponds to approximately 18% spillover (PE-%FITC).Ĭlick here for instructions on modifying the matrix. Thus, 0.1803 is the spillover coefficient between FITC and PE, or the amount of FITC signal in the PE channel for a FITC-stained cell 0.0091 is the amount of the PE signal in the FITC channel for a PE-stained cell. The rows are the spectra of each channel the columns are the contributions of each fluor into the channels. The subsequent lines are the spillover coefficients, corresponding to each of the channels named. The names must match exactly use the names that appear in the $PnN keywords of the FCS file. Channels that are not in this list are not compensated channels in this list that are not in the sample are ignored. The second line has the names of the fluorescence channels that are being compensated. The first line has the title of the compensation matrix. If you save the matrix to a file (using the 'Edit/Save Matrix' option), the file looks like this: CompMatrix

When the 'Compute' button in the compensation matrix dialog window is clicked, FlowJo generates a compensation matrix.