Modfit lttm software

A cell cycle PI staining assay was performed to determine the effects of miR-149-5p oligonucleotides, pcDNA3.1(+)-miR-149-5p, CMTM3-OE, and CMTM3-shRNAs on the different phases of the cell cycle in hair follicle stem cells. After cells were transfected for 48 h, they were collected in 6-well plates and centrifuged at 1500 rpm/min for 5 min. The supernatant was discarded after centrifugation, and the pelleted cells were washed once with 1 ml of precooled 1 × PBS. Afterward, cells were incubated with 1 ml of precooled 70% ethyl-alcohol at 4°C for 12 h. Subsequently, the cells were resuspended in 500 μl of PI staining buffer (Beyotime, Shanghai, China) and incubated at 37°C for 30 min in the dark. Then, the cell suspensions were subjected to flow cytometry analysis (FACSAria SORP, BD BioSciences, NJ, United States). ModFit LT^TM software (Verity Software House, Topsham, ME, United States) was used to create and analyze the cell cycle histograms.

The percentage of apoptotic nuclei was measured by propidium iodine (PI) staining as previously described (Nicoletti et al., 1991 (link)). THP-1 cells were infected with pneumococcus at an MOI of 30 and 100 for 4.5 h, followed by fixation with 70% cold ethanol at -20 °C overnight. The fixed cells were stained with PI solution (PBS containing 20 μg/ml PI (Sigma-Aldrich), 400 μg/ml RNase A (Sigma-Aldrich), and 1% Triton X-100) for 30 min at room temperature in the dark. The PI fluorescence of individual nuclei was measured by FACS Calibur and analyzed with DNA analysis ModFit LT^TM software (Verity Software House).

After 24 h following HNK (25 µM) treatment, the cells were harvested and suspended in PBS containing 0.5% BSA for 10 min at room temperature, followed by the addition of 10 μL phycoerythrin-conjugated DCLK1 antibody (Abcam Inc, Cambridge, MA, USA) at room temperature for 1h dark in a rotator. The samples were analyzed using a FACS Calibur analyzer (Becton Dickinson, Mountain, View, CA, USA), capturing 10,000 events for each sample. The results were analyzed with ModFit LT TM software (Verity Software House, Topsham, ME, USA).

The HCT 116 cells were seeded in 6-well plates (Nunc, ThermoFisher, Waltham, MA, USA) at a density of 5 × 10⁴ cells/well. After 24 h the medium was replaced with tested compounds. The cells were incubated for 24 h at 37 °C. Afterward, floating cells were collected and adherent cells were harvested by trypsinization and centrifuged at 600× g. The cells were washed with PBS (phosphate buffered saline) (Sigma-Aldrich, Taufkirchen, Germany) and then fixed in 70% ice-cold ethanol and stored at −20 °C overnight. The next day the cells were centrifuged at 600× g, washed with PBS, treated with 100 μg/mL RNase A solution (EURx, Gdańsk, Poland), 100 μg/ml solution of propidium iodide (PI) (Acros Organics, Geel, Belgium) and incubated for 30 min at room temperature in the dark. After staining, the cells were analyzed using a Becton Dickinson FACS Aria III sorter (BD Company, San Diego, CA, USA). Experiments were repeated at least twice. Data were analyzed using ModFit LTTM Software (Verity Software House, Topsham, ME, USA).

1 x 10⁵ cells per well were seeded on a 6-well plate in 3 ml media and treated with different concentrations of AGI-5198 (0, 1, 5, 10, 20 μM) or 20 μM DMSO for 72 h. Apoptosis was detected using Annexin V-FITC kit (Beckman Coulter, France) according to the manufacturer’s instructions. Cells stained with Annexin V-FITC and propidium iodide (PI) were analyzed by LSR-II flow cytometry (Becton Dickinson, USA). Apoptotic cells were quantified, including early apoptotic cells only positive for Annexin V and late apoptotic cells double positive for both Annexin V and PI. Data was analyzed by FlowJo-V10 software (FlowJo, USA).
For cell cycle analysis, non-treated or AGI-5198-treated cells were resuspended in 500 μl cold PBS. Cells were then fixed by addition of 4.5 ml 70% cold 200-proof ethanol (Sigma, USA) for at least 14 h at -20°C. The fixed cells were then spun down at 300 g for 10 min at 4°C and washed once using cold PBS. Cells were stained with propidium iodine with RNase (Cell Signaling Technology, USA) and analyzed using LSR-II flow cytometry. Data was analyzed by ModFit LTTM software (Verity Software House, USA).