Team:William and Mary/Flow Protocol

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Preserving Cells for Flow Cytometry

While single cell fluorescence measurements via flow cytometry are one of the more rigorous and useful methods of characterization in synthetic biology, many teams, especially teams from smaller schools are unable to perform flow cytometry measurements due to lack of access to a cytometer. This is not surprising given that instruments are quite expensive to purchase (often $50,000 or more) and maintain. Even for teams with access to an institutional flow cytometer, their use comes with significant cost ($80-$100 an hour). This issue is particularly evident during time course measurements, as experiments often last multiple hours, which means that a single flow cytometry experiment live cell imaging could potentially cost hundreds of dollars. Further, based on our own experiences we knew that time course flow cytometry was labor intensive in part due to our desire to immediately measure the samples after time points were taken (as previous experience had shown that fluorescence values are not stable for long periods of time at room temperature). To solve this problem, we implemented a method that utilizes freezing E. coli at -80C for long term preservation, allowing for samples to be collected one day and measured another, as well as to be sent (on dry ice) to another institution/team for measurement.
The procedure is simple: upon taking a measurement sample, immediately place the sample on ice (in our experience iced sample's fluorescence as measured by FACS is stable for at least 40 minutes), add glycerol to a final concentration of 15% (we recommend the use of 87% or less glycerol to enable quick mixing), mix, and then freeze via liquid nitrogen or by placing sample at -80C. To measure, thaw samples on ice and then filter into cold FACS buffer or PBS, diluting by at least 10 fold and keeping diluted samples on ice until measurement is complete. In our experience, these methods work quite well and sample's fluorescence are stable for at least two weeks.
In order to show that our protocol does not significantly alter the final values of samples we performed a validation by using the ATc inducible circuit BBa_K2333428. In brief, we grew samples overnight, diluted, grew for 2 hours and induced with 50ng/mL ATc, taking time points every 20 minutes. Half of the culture from each time points was flash frozen in liquid nitrogen and half was immediately measured on the flow cytometer. The frozen samples were stored for 3 days at -80C, before being measured. See the data and methods page for more details and raw data. The result of this validation experiment shows that there is little if any difference between the measurement values of the frozen and nonfrozen cells (Figure 1).
Figure 1: Single cell fluorescence measurements of BBa_K2333428 time course live (NonFrozen) and 3 days later (Frozen). Each dot represents the geometric mean of at least 10,000 single cell measurements each of 3 biological replicates, the shaded region represents one geometric standard deviation above and below the geometric mean.