Team:NCTU Formosa/protocol F

Protocols

Flow cytometry

1. Principle :

Flow cytometry is a technology that simultaneously measures and then analyzes multiple physical characteristics of single particles, usually cells, as they flow in a fluid stream through a beam of light. The properties measured include a particle’s relative size, relative granularity or internal complexity, and relative fluorescence intensity.

These characteristics are determined using an optical-to-electronic coupling system that records how the cell or particle scatters incident laser light and emits fluorescence.

A flow cytometer is made up of three main systems: fluidics, optics, and electronics(Figure 1).


1. The fluidics system transports particles in a stream to the laser beam for interrogation.
2. The optics system consists of lasers to illuminate the particles in the sample stream and optical filters to direct the resulting light signals to the appropriate detectors.
3. The electronics system converts the detected light signals into electronic signals that can be processed by the computer. For some instruments equipped with a sorting feature, the electronics system is also capable of initiating sorting decisions to charge and deflect particles.

Figure 1. Sorting components on the FACSVantage SE



In the flow cytometer, particles are carried to the laser intercept in a fluid stream. Any suspended particle or cell from 0.2–150 micrometers in size is suitable for analysis. When particles pass through the laser intercept, they scatter laser light. Any fluorescent molecules present on the particle fluoresce. The scattered and fluorescent light is collected by appropriately positioned lenses. A combination of beam splitters and filters steers the scattered and fluorescent light to the appropriate detectors. The detectors produce electronic signals proportional to the optical signals striking them. List mode data are collected on each particle or event. The characteristics or parameters of each event are based on its light scattering and fluorescent properties. The data are collected and stored in the computer.




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2. Materials and Methods :
   1. Host Cell

      TransforMax EPI300 E. coli cells from Epicentre Technologies (Madison, WI) were used for the fluorescence measurable experiments. DH5α E.coli cells from Yeastern biotech (ECOS) were used for all constructed procedure.

   2. Growth Conditions and Measurements

      Bacteria were grown in 14-ml tubes at 37°C incubator, shaking at 150 rpm, with LB (Luria-Bertani) medium (50.0 mg Tryptone, 25.0 mg Yeast extract, 50 mg NaCl, and 5 ml ddH2O) and proper antibiotics in plasmid constructed step. For flow cytometry analysis, bacteria were grown in 14-ml tubes at 37°C incubator, shaking at 150 rpm, with M9 minimal medium (33.9 mg Na2HPO4, 15 mg KH2PO4, 2.5 mg NaCl, 5 mg NH4Cl, and 5 ml ddH2O) which supplemented with 0.2% casamino acids, 1 mM MgSO4, 2 M thiamine, 10 mM glucose, and proper antibiotic (25 ng/ml Kanamycin, 10 ng/ml Tetracyclin). After overnight growth, 5 μl from 12 cultures which contained different expression devices were transferred into flesh medium and GFP fluorescence intensity were collected by flow cytometry (CyFlow from Partec.) In the repressor-controlled genetic circuits, cell density grew to a O.D. 600 (optical desity at 600 nm) of 0.1 (equal to 1×108 cells per ml) in 14-ml tubes containing 5 ml M9 minimal medium, then 1000 ng/ml Anhydrotetracycline (aTc) was added to the medium. After adding aTc inducer, seven hours of continuous measurement of a point every fifteen minutes by flowcytometry (CyFlow from Partec.) to achieve time-course data collected [20]. In dose depended data, when cell density grew to a final O.D. 600 of 0.1 (1×108 cells per ml) in 14-ml tubes containing 5 ml M9 minimal medium, then different concentration aTc (0 ng/ml, 50 ng/ml, 100 ng/ml, 150ng/ml, 250 ng/ml, 500 ng/ml, 1000 ng/ml, 2000 ng/ml) was added to the medium. After seven hours, fluorescence intensity of GFP was measured by the flowcytometry (CyFlow from Partec.). In addition to all fluorescence measurements were performed on the flow cytometer equipped with a 488 nm argon excitation laser and a 515–545 nm emissions filter. For each sample, 10,000 events were collected. Fluorescence intensities were converted to Molecular Equivalents of Fluorescent (MEFL) based on daily measurements of SPHERO Rainbow Calibration Particles (Spherotech, Libertyville, IL)

   3. Data Analysis, Computational Models, and Simulations

      Fluorescence from an individual sample was calculated using CyFlow software (Partec.). The original log-binned fluorescence intensity values were collected with cell number counting, then, the normal distribution fluorescence intensity graphics could be ranged for obtaining mean and standard deviation of the resulting values. The mean and standard deviation of the resulting values were obtained for each sample within a small forward and side scatter gate to reduce variability in cell size and shape. Finally, the mean of fluorescence intensity will be transformed into Molecular Equivalents of Fluorescent (MEFL) by the conversion of standard curve of measurement of SPHERO Rainbow Calibration Particles (Peak Technology Co., Ltd.). Computational models were developed based on chemical mass action kinetics, and the resulting analytical formulas were fitted in Matlab (Math-Works) to the average of 3 experimental replicates.

   
   
   
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   Figure 4.
   

   (CyFlow from Partec.)

Reference
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