Team:BostonU/FlowCytometry

Here, we describe how we used BBN Technologies TASBE tools for analyzing our flow cytometer data obtained during the Test part of our Chimera Workflow. To view our experimental results, please check out our Data Collected page.

Using BBN Technologies TASBE tools, we were able to quickly and easily analyze the flow cytometry data obtained for our various devices. We collected data using a BD LSRFortessa outfitted with a high throughput sampler, allowing for the fast capture of 50,000 cells per sample in a 96-well format. Since we focused on using green fluorescent protein (GFP) and red fluorescent protein (RFP) for our reporter proteins, we utilized the 488nm blue laser with a 530/30 filter and a 561nm yellow green laser with a 610/20 filter, respectively. The GFP protein is excited by the 488nm laser and emits light that will be collected by the 530/30 bandpass filter, while the RFP is excited by the 561nm laser and emits light that will be collected by the 610/20 bandpass filter.

Below, you can see more details of our experimental design and the controls we used, which are required by the TASBE tools in order to convert the arbitrary fluorescence units obtained from the flow cytometer into absolute units in the form of molecules of equivalent fluorescein (MEFL). This allows the user to show their data in absolute units that then allow scientists to compare experiments across labs and machines.

As a part of these controls, we used Spherotech's 8-peak particles (RCP-30-5A) in order to obtain standard MEFL units for the FITC channel. They are also used to measure the long term performance of the flow cytometer and are included in every experiment run through the flow cytometer.

The Cytometer Setup and Tracking beads offered by BD Biosciences were also utilized to set the laser delay and optimize the cytometer settings 15-30 minutes prior to running any samples through the Fortessa.

In order to obtain MEFL measurements for the RFP protein, we had to utilize a dual positive control that had a FITC channel fluorescent protein and a Texas Red channel fluorescent protein. We used GFP for our FITC control and RFP for our Texas Red control. We used the MoClo versions of the J23014 promoter in both devices, E1010 for RFP and E0040 for GFP, along with the BCD2 5'UTR element and B0015 terminator. For the two color controls, we built them with the same parts, with RFP in the first transcriptional unit and GFP in the second for one control and vice versa for the other control (shown above in Controls figure).