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FACS sorting
This protocol is extracted from research article:
Triangular correlation (TrC) between cancer aggressiveness, cell uptake capability, and cell deformability

Procedure

Generation of two subcell populations originated from a single cell line, differing by their capacity to uptake 2.4-μm beads, was done by the FACS sorter. Each FACS sorting procedure separated cells that did not uptake any particles (negative, FL2Low) from the rest of the cells (positive, uptake of at least one bead, FLhigh). Therefore, after the first sorting, in each cycle, we obtained four different subsets of cells: positive-positive; positive-negative, negative-positive, and negative-negative. Only the positive-positive and negative-negative were kept for further sorting. In each cycle, the sorting enrichment, Δ (presented in Fig. 4C), was defined as $Δ≡J++−J+−$, where $J++$ was the percent of positive cells obtained in a sorting procedure on the positive-positive population of the previous cycle and $J+−$ was the percent of positive cells obtained in a sorting procedure on the negative-negative population of the previous cycle. An exception to this definition is the first cycle where both of the percent values of positive cells were calculated relative to the same origin population of cells, yielding $J++−J+−=0$. Each sorting included the flowing steps. Cells were cultured for five generations and incubated with 2.4-μm beads for 24 hours. Cells were then washed thoroughly with PBS and detached with trypsin, centrifuged, counted, and diluted to 1.0 × 107 cells/ml in PBS. Using a BD FACSAria III cell sorter, cells were analyzed and sorted on the basis of the fluorescence intensity distribution (FL-2). The levels of fluorescence intensity indicated accurately on the number of internalized particles, as shown in fig. S2, allowing for a highly sensitive uptake-based sorting.

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