Facs lsr 2

For HDR at AAVS1 and LMO2, 293T cells were transfected with Lipofectamine 3000 (Thermo Fisher Scientific) and the appropriate SpCas9 and SpCas9-DN1S gRNA-containing plasmids, and donor template plasmids with homology arms for LMO2 or AAVS1 with a self-promoted GFP cassette. Transfection efficiency was assessed by BD FACS LSR II at 72 h post-transfection. At 15 days post-transfection, GFP+ events were recorded by BD FACS LSR II to determine the percentage of HDR events. %HDR reported was normalized to the transfection efficiency, or percentage of donor and Cas9 double positive cells collected at 72 h post-transfection.
For HDR at the CD45 locus, K562 cells were electroporated with SpCas9 or SpCas9-DN1S plasmids containing the CD45 gRNA plus a CD45-GFP plasmid donor using the Neon^TM Transfection System (Thermo Fisher Scientific). Cells were sorted for the double positive Cas9 and donor population 48 h after electroporation. Later, the cells were checked for GFP and CD45 (PE-Cy7, BD Biosciences 557748) expression by a BD LSR II instrument. HDR was determined by the %GFP+ CD45+ population.

The SP protocol was essentially performed as described by Goodell et al [25] (link). Cells (1×10⁶ cells per ml) were incubated in Dulbecco modified Eagle's medium containing 2% heat-inactivated FBS, 10 mM Hepes and 5 µg/ml Hoechst 33342 (Sigma) for 120 min at 37°C in the absence or presence of verapamil (50 µM). Propidium iodide was added to discriminate dead cells. The SP population was identified and sorted by its fluorescence profile in dual wavelength analysis (450/20 and 675/20 nm) after excitation at 350 nm. Samples were analyzed by flow cytometry using a FACS LSRII (BD Biosciences). For the determination of CD44/CD24 phenotype, cells were washed with phosphate-buffered saline (PBS), detached with accutase treatment and re-suspended in PBS supplemented with 0.5% BSA. Combinations of fluorochrome-conjugated monoclonal antibodies against human CD44 (FITC) and CD24 (APC) were obtained from Beckman Coulter. Specific antibodies or the respective isotype controls were added to the cell suspension, as recommended by the manufacturer, and incubated at 4°C in the dark for 20 min. Cells were washed with PBS containing 0.5% BSA, centrifuged and re-suspended in PBS with 2% paraformaldehyde. The labeled cells were analyzed on a FACS LSR II (BD Biosciences).

For cell cycle analysis, HeLa cells were harvested and washed with PBS. For fixation and staining the pellet was resuspended in DNA staining solution I (10 μg/ml RNase, 0.6 mg/ml NaCl, 1 mg/ml Sodium citrate, 0.07% NP-40, 10 μg/ml propidium iodide (PI) in PBS). After incubation for 30 min at RT, DNA staining solution II (15 μg/ml citric acid, 85 μg/ml sucrose, 10 μg/ml PI in PBS) was added. Samples were stored at 4°C until cell cycle data were collected with FACS BD LSR II (Becton Dickinson) and analysed with the free flow cytometry software FlowPy.
For DSB-repair reporter analysis, HeLa pEJ and HeLa pGC cells were harvested 48 h after transfection of pMCV-I-SceI. The cell pellet was washed twice with PBS before fixation with a solution consisting of 3% PFA and 2% glucose in PBS for 10 min on ice. After fixation, PBS was added and cells were washed one more time with PBS. Samples were stored at 4°C in PBS with 10% FBS until measurement of GFP-positive cells with FACS BD LSR II.