Nexcelom cellometer

Gene-edited human HSPCs and controls were cloned in 96-well tissue culture plates by limiting dilution. Briefly, electroporated cells were stained with acridine orange-propidium iodide (AOPI) to determine viability and enumerated using a Nexcelom Cellometer (Nexcelom Bioscience, Lawrence, MA, USA). Viable cells were then diluted in StemSpan SFEM II tissue culture medium (supplemented with 100 U/mL penicillin; 100 µg/mL streptomycin; and 100 ng/mL of TPO, SCF and Flt3-L) to a concentration of 1 cell per 200 µL medium. Diluted cells were distributed into 96-well tissue culture plates (200 µL medium per well). Individual wells were inspected by bright-field microscopy to confirm the presence of a single cell. Ninety-six well plates were incubated in a humidified 37 °C, 5% CO₂ incubator under normoxic conditions for up to 4 weeks prior to genomic DNA isolation.

Following control and six days of sim µg exposure, the cell diameter was analyzed using a Nexcelom Cellometer (Nexcelom Bioscience, Lawrence, MA, USA). Two independent experiments were performed and a total of 513 cells were analyzed for the control, as well as 362 for adherent cells and 349 for the non-adherent cells. Cells were binned based on size and significant changes with sim µg exposure defined.

Cell line pools were made in sets of 25 cell lines. These 25 cell line pools were chosen based on doubling time and were grown in RPMI without phenol red and with 10% fetal bovine serum (FBS). Cell lines were then washed with 10 mL of phosphate-buffered saline (PBS) and trypsinized with 1 mL trypsin, which was then removed. Ten milliliters of RPMI media were added to the cells post trypsinization and resuspended. Cells were then counted by a Nexcelom cellometer using 10 µL of cell suspension and 10 µL of Trypan blue. Equal numbers of cells per cell line were mixed together and spun down at 1250 rpm for 5 min. Media was aspirated and the cells were resuspended in Sigma Cell Freezing media and frozen in 1 mL aliquots. This process was repeated for all of the 25 cell line pools. For MIX-Seq experiments involving larger pools, multiple 25 cell line pools were thawed in RPMI with 10% FBS, spun down, and resuspended in 5 mL of RPMI media. Cells were then counted and equal numbers were combined together on the day of plating to form larger pools of up to ~100 cell lines.