Team:BostonU/FlowCytometry

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 cells in a 96-well format.

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.

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

We also 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 should be included in every experiment run through the flow cytometer.

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 Characterization Workflow figure).