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By FACS Canto II FACS measurements can be carried out independently or by the core facility staff. The device is equipped with the following lasers:

• 407 nm: violet laser
• 488nm: Blue Laser
• 633 nm: Red Laser

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1. Cells are harvested by a sheath flow as beads strung on a string (hydrodynamic focusing)

2. In the laser intersection the analysis of the cells takes place.

3. The nozzle separates the jet into individual drops and charges the drops to be sorted on.

4. The charged drops are deflected in an electric field, and fall into the appropriate receptacle.

The following tables show how long a sorting operation. These are minimum hours, 20 minutes should be allocated for the setting of the device. The duration varies with the size of the cells.

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Examples for multicolor analysis

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1) separated into 2 populations in 15 ml tubes

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2) seperated into up to 4 populations in FACS-tubes or small reaction tubes

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3) filing in microtiter plates (for example, 96-well)

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This Cytometry one or more cell populations from the suspension can be sorted out (Note: not all cells can be sorted well, sometimes the populations are very sensitive). The sorted cells can be cultivated further.
The cell sorting on FACS AriaIIIu is handled by employees of the Core Unit. The device is equipped with the following lasers:

• 407 nm: violet laser
• 488nm: Blue Laser
• 561 nm: green laser
• 633 nm: Red Laser

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After acquiring your samples at the flow cytometer and manual gating of the cell populations of interest in FlowJo or Diva, you end up with a (huge) Excel table containing all samples and all cell populations. Creating t-SNE plots and heatmaps from this table gives an overview of your data and points out subgroups of samples with similar immunophenotype. We can also include clinical parameters and sample metadata into this kind of analysis.

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After acquisition, proper compensation and removing trash and unwanted cell populations from your FCS files, this analysis creates t-SNE plots directly from your FCS files. The plots will be colored by expression of all markers included in your panel and by density, resulting in a single-cell overview of your data showing all cell populations you might already know plus maybe cell populations you have not been looking at so far. It is possible to create t-SNE plots for visually comparing samples or sample groups.

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Just like for analysis type (2), data has to be properly compensated and trash and unwanted cell populations have to be removed from your FCS files. This analysis is then capable of dividing the cells into clusters with distinct phenotypes (comparable to what manual gating in FlowJo or Diva is doing) in an unsupervised manner, resulting in a SPADE tree. These trees can further be analyzed and thereby groups of samples can be compared revealing cell clusters being differentially abundant between the groups.

Data quality is essential for this kind of analysis, therefore we advise you to contact us prior to conducting your experiment so that the experiment and data acquisition can be planned in an appropriate manner.