Tc10 cell counter

EPS-blastoids were manually picked up using mouth pipette and washed three times in PBS containing 0.04% BSA. Around 500 EPS-blastoids were harvested and dissociated with a homemade enzyme mix composed of 0.5X versene (Lonza, 17711E), 0.5X Acumax (Innovative Cell Tech, AM105), and 0.05X Dnase (STEMCELL Technologies, 07900) at 37°C for 30min with agitation. Dissociated cells were spun down and wash with PBS + 0.04%BSA for three times and resuspended in the same buffer. Cell density was determined by a TC10 cell counter (Bio-Rad, 1450001). Blastocysts were dissociated using the same protocol. Dissociated cells (~4800 cells for EPS-blastoids and ~1000 cells for blastocysts) were loaded into the Chromium Single Cell B Chip (10X Genomics, PN-120262) and processed in the Chromium single cell controller (10X Genomics) to generate single-cell gel beads in the emulsion according to the manufacturer’s protocol. The library was generated using the Chromium Single Cell 3’ Reagent Kits v3 (10X Genomics, PN-1000092) and Chromium i7 Multiplex Kit (10X Genomics, PN-120262) according to the manufacturer’s manual. The two libraries were pooled and sequenced using Nextseq 500 (150 cycles, high output).

Cortical organoids were dissociated into single-cell suspension using Accumax (Sigma Aldrich) for 30min at 37°C with rotation (95 rpm). Then, organoids were disaggregated using a 1000μl pipette tip, incubated for another 10min at 37°C in suspension with rotation (95 rpm), and centrifuged for 3 minutes at 100xg. The cell pellet was resuspended in Media2 containing 5 μM of ROCK inhibitor, filtered through a 100μm mesh (Gibco) and centrifuged again for 3 minutes at 100xg. To further remove un-dissociated organoid tissue, the procedure was repeated but with filtering through the 40μm mesh (Gibco). Cells from suspension were counted using a Bio-Rad TC10 Cell Counter.

Cell growth inhibition assay was performed as described previously.^{34 (link)} In brief, 0.5 million cells per well were seeded in triplicate into 24-well plates, subjected to treatment with various final concentrations of compound. Fresh medium containing compound was changed every 2 days. All flow-growing cells were periodically diluted to keep the cell density less than 1 × 10⁶/mL. Cell numbers were counted by an automated TC10 cell counter (BioRad) every 2 days.

Cell growth inhibition assay was performed as described previously (Xu et al., 2015 (link)). In brief, 0.5 million of cells per well were seeded in triplicate into 24-well plates, subjected to treatment with various final concentrations of compound. Fresh medium containing compound was changed every two days. All flow-growing cells were periodically diluted to keep the cell density less than 1×10⁶/mL. Cell numbers were counted by an automated TC10 cell counter (BioRad) every two days. Effective control to 50% growth inhibition (EC50) values were calculated via a nonlinear regression analysis by using data from at least three experiments and presented as the mean ± SD.