Moflo high speed cell sorter

For cell cycle analysis, 0.5 × 10⁶ cells in 100 μL buffer (PBS + 0.5% BSA) were stained with PI and assessed by flow cytometry according to standard protocols. For analysis of apoptosis, 0.25 × 10⁶ cells were collected on day 6 and day 7 of culture and resuspended in 50 μL buffer. The cells were then stained with Annexin V and 7AAD according to the manufacturer's protocol. To monitor erythroid progenitor differentiation, 0.25 × 10⁶ cells in 50 μL buffer were stained with 7AAD, GPA, IL3R, CD34 and CD36. To monitor erythroid terminal differentiation, 0.25 × 10⁶ cells in 50 μL buffer were stained with 7AAD, GPA, Band3 and α4 integrin. For enucleation, 1 × 10⁶ cells were collected on day 13 and day 19 of culture and resuspended in 200 μL buffer (PBS + MgCl₂). The cells were then stained with Syto‐16 and 7AAD according to the manufacturer's protocol (BD Pharmingen). Stained cells were analysed with a FACS Canto II (BD Pharmingen), and all data analysis was performed by FlowJo and BD FACSDiva software. For cell sorting, erythroid progenitor CFU‐E cells were stained as described above and sorted on a MoFlo high speed cell sorter (Beckman‐Coulter).

At the time points indicated, 1.10⁶ vector-transfected or infected cells were trypsinized and washed twice in phosphate-buffered saline (PBS) prior to fixation with 70% ethanol at 4°C for 24 h. Cells were then washed twice in cold PBS and incubated in PBS containing 500 µg/ml Ribonuclease A (Sigma-Aldrich) at 37°C for 1 h. After filtration through a 30-µm pore size membrane, cells were stained with 10 µg/ml propidium iodide (Invitrogen) for 15 min in the dark. Flow cytometry analysis was performed using a MoFlo high-speed cell sorter (Beckman Coulter, Fort Collins, CO, USA) equipped with a solid-state laser operating at 488 nm and 100 mW. Cellular DNA content was analyzed with a 740 nm long-pass filter. Doublets were discarded on the basis of combination of pulse width and area/peak fluorescence. eGFP autofluorescence was detected with a 530/40 nm band-pass filter and the cell cycle distribution was specifically analyzed for eGFP-positive versus eGFP-negative cells. Cell cycle profiles were analyzed with the MultiCycle AV software (Phoenix Flow Systems, California, USA).

Freshly isolated cells were seeded on collagen IV-coated plates for 24—36 h to revive CD44 expression diminished by digestive enzymes. Both unattached and attached (trypsinized) cells were collected and blocked with 1% BSA, 4% normal goat serum in PBS. Cells were stained with AF488-EpCAM (BioLegend), APC anti-human CD44 (BioLegend), and their respective isotypes. Cells were sorted using a Beckman Coulter MoFlo high-speed cell sorter. Unstained, isotype, and single-color controls were performed. The gates for CD44 and EpCAM were set based on the results of isotype, and single-color controls were run in parallel. The results were analyzed using FlowJo 10.1.

HeLa cells were transduced with 200 ng CAp24 of lentivirus prepared as described above. After 24 h transduction, cells were washed, replaced with new culture medium and further incubated for 48 h. At 72 h post-transduction, cells were trypsinized, filtered through 37 μm Nylon Mesh to remove cellular clumps and diluted in PBS to a concentration of 2 × 10⁷ cells/ml. Fluorescence-activated cell sorting (FACS) analyses were performed to isolate cells expressing high levels of mCherry using a MoFlo high speed cell sorter (Beckman Coulter). The isolated HeLa cells expressing Nullbasic-mCherry (Hela-Nullbasic-mCherry) or FLAG-mCherry (Hela-FLAG-mCherry) were expended and prepared for proteomic experiments.

Percentage of dead cells was determined by staining with propidium iodide (PI 1 μg/ml eBioscience) and acquisition was performed on flow cytometry (Canto, BD Biosciences). Analysis was performed using FlowJo software. In some experiments GFP positive population of SK-N-MC cells transfected with pEGFP and pEGFP-DHX9 constructs, was isolated using a MoFlo high speed cell sorter (Beckman Coulter).

For ChIP experiments, epiblast cells were isolated from E6.25 pre-gastrulating embryos coming from outbred MF1 females crossed with GGOF stud males. After recovering embryos from the decidua, Reichert’s membrane was dissected out and extraembryonic cone was also removed. Remaining tissue was used to prepare single cell suspension for sorting using MoFlo high-speed cell sorter (Beckman Coulter, CA) based on EGFP expression. The purity of epiblast cells was assessed by staining for SOX2 and was in excess of 95%. For single epiblast experiments, embryos were dissected as previously described (Tesar et al., 2007 (link)).