Cytometer.docx

Several research programs here are BARC are presently or evolving to perform more sophisticated cell biology research. Especially in that blood as well as dispersed tissues contain a myriad of different types of cells, the capabilities of defining the exact phenotypic characteristics and activities of the different types of cells is totally hampered unless purified populations can be obtained out of this complex cell mixture and further either propagated or research as a pure cell type. More specifically, there is a tremendous need to develop cell models for both swine and poultry applications, capabilities that really do not exist past the elementary principles of primary culture. The issue with primary culture is that you are still faced with performing experiments on a mixed populations and types of cells. Fluorescence cell sorting and capture is the state-of-the-art process for obtaining these otherwise difficult to harvest purified cells and subpopulations of cells for these food producing species. This is extremely important towards the exact application of gene editing technologies whose effects can be investigated by in vitro models without the need to generate transgenic animals.

Because of the complex nature of how circulating immune cells interact and in light of the number of combination changes in cell population types can potentially change in response to an infection over time, our needs require that up to 9 different cell populations be able to be distinguished in a single rum.

The instrument required is a multichannel fluorescence flow cytometer. The instrument is capable of differentiating up to 9 different populations of cells in a mixed culture, isolating the different cell populations and capturing and placing the different cell populations each into its own separate collection vial. Cells isolated in this manner are highly viable, capable of being further cultured and used to determine specific unique characteristics of the identified and isolated types of cells. By being able to sort and collect cells, specific cell-to-cell interactions can be studied, presently a capability nor present at BARC.

The Instrument includes the Cell sorter, the fluidic cart, the monitor and computer, the ACDU (plate sorting, 6 well -384 well), 3 laser, 9 colors, 11 parameters.

Other salient features include:

•Sensitivity, published MESF values for FITC <80 and <30 for PE.

•QBr values (Fluorescence Detection Efficiency, published) FITC Qr (x1000): 40 photoelectrons/ABD PE Qr (x1000): 325 photoelectrons/ABD

Numerical aperture 1.2. Increases sensitivity •Droplet sorting

•Purity and yield At 23 psi and 34 kHz with an average of 10,000 events per second, a two-way sort of 5% target populations achieved a purity of 98% and a yield of >80% of Poisson’s expected yield for both populations. No limitation is imposed on the event rate (one event constitutes a selected cell being gated out of the general population, counted and collected in a receptacle vessel). However, yield decreases with higher event rates based on Poisson distribution principles.

•Sort collection

•PCR tray, microscope slide (optional)