Click it edu incorporation kit

Cells were fixed in 4% paraformaldehyde and permeabilized in 0.1% Triton X-100 before blocking in 10% normal goat serum/1% BSA (Millipore Sigma). Cells were incubated with primary antibodies (α-actinin [1:200; A7811; Sigma-Aldrich], cardiac troponin T [1:100; ab45932; Abcam], KI67 (1:200; ab8330; Abcam], PHH3 [1:200; 9701; Cell Signaling Technology], and Aurora B kinase [1:100; ab2254; Abcam]) overnight, and detection was mediated by incubation with secondary antibodies (Alexa Fluor antibody conjugates; Thermo Fisher Scientific) for 1 h. DAPI was used as a nuclear marker. Mounting was performed using Fluoromount-G mounting medium (Thermo Fisher Scientific).
To determine cell loss via pathways involving DNA fragmentation, TUNEL assays (Roche) were performed according to the manufacturer’s instructions. In vitro proliferation after FSTL1 supplementation was assessed using Click-iT EdU incorporation kit (Life Technologies). Imaging was performed using a confocal microscope (Leica SP8 X) and image analysis using ImageJ.

For apoptosis analysis, flow cytometric determination of annexin V binding, mitochondrial membrane potential loss (Δψ_m), conformational change in BAX and caspase-3 cleavage were carried out.^{29 (link)} For gating stem/progenitor population (CD34⁺CD38^low/− cells) in primary AML samples, anti-human CD34 and CD38 antibodies (BD Biosciences, San Jose, CA, USA) were used. Cell cycle distribution was analyzed by the Click-iT EdU incorporation kit (Life Technologies). Staining of intracellular BMI-1 and p53 was performed as described previously.^{19 (link), 30 (link)} Expression level was measured as the mean fluorescence intensity ratio (MFIR) calculated by the formula: MFIR=(MFI for specific antibody)/(MFI for isotype control).

Cell cultures were supplemented with 10 μM EdU for 14 h, harvested and EdU incorporation was detected by the FACS-based Click-it EdU incorporation kit (Life Technologies) according to the manufacturer's instructions.
Co-culture: 2 × 10⁵ cells/well NB-cells were seeded in 12 well plates as single cultures or were mixed at a ratio of 1:5, 1:2 and 2:1, resp. Medium was replenished every 3 days and cells were passaged, if required. 1 week after culture start, cells were analyzed for cell cycle state by EdU incorporation. Cells were additionally stained with m-anti CD44-PE and anti GD2-FITC and acquired using the BD FACS Fortessa and the Diva software (BD).