Live-cell imaging of meiotic cells

Images were acquired using a DeltaVision Elite wide-field fluorescence microscope (GE Healthcare) and a PCO Edge scientific complementary metal–oxide–semiconductor camera, with softWoRx software and a 60× NA1.42 oil-immersion Plan Apochromat objective. GFP and RFP filter sets were used, with the setting information for acquiring each figure panel provided in Table S4. Live-cell imaging was performed exactly as described in King et al. (2019) (link), except fresh SPO was used in place of conditioned SPO. In short, cells were imaged in an environmental chamber heated to 30°C, using either the CellASIC ONIX Microfluidic Platform or concanavalin A–coated glass-bottom 96-well plates. Cultures of meiotic cells in SPO were transferred to the microfluidic plates and loaded at 8 psi for 5 s. SPO was applied with a constant flow rate pressure of 2 psi for 15–20 h. With plates, cells were adhered to wells, and 100 µl of SPO was added to each well. Specific imaging conditions are noted in Table S4. All time-lapse experiments were performed using the CellASIC system (EMD Millipore) in Y04D or Y04E microfluidics plates, with the exception of the LatA experiments, for which we used glass-bottom 96-well plates (Corning). Images were deconvolved using softWoRx software (GE Healthcare) using 3D iterative constrained deconvolution with 15 iterations and enhanced ratio.