- Prepare cells for FACS analysis as described in Subheading3.3,
steps 13– 19 for suspension and adherent cells. - Set up the flow cytometer with negative control cells. Adjust
the forward scatter (FSC) and side scatter (SSC) to place the
population of interest on scale. - Run positive control cells, and draw an interval gate to define
the populations of interest. - Once gates have been determined, sort the top ~10% of
fluorescent-positive cells from the experimental samples.
Fig. 3(continued) detection. Single-cell sgRNA AMPKα1 Caco2 clones were selected based on puromycin
resistance (left part), and then single-cell sgRNA AMPKα2 Caco2 clones on a AMPKα1 knockout background
are isolated based on GFP fluorescence (right part). (B) Genome editing efficiency of AMPKα2 sgRNA in
AMPKα1-knockout Caco2 cells is assessed by capillary electrophoresis fragment analysis. (C) Western blot
analysis of AMPKαprotein expression in single-cell Caco2 clones isolated after sequential transduction with
lentivirus expressing “puromycin” CRISPR/AMPKα1 and “GFP” CRISPR/AMPKα2. Western blots were per-
formed using anti-panAMPKαand anti-β-actin antibodies. Each clone was analyzed in triplicate
184 Adrien Grenier et al.