Cytoflex 5

For 4-dpf fish, 100 embryos were collected and washed once in ice-cold PBS. We removed all the solution into a 50-mL Falcon tube, then passed it through a 100-µm nylon filter (BD Falcon, Cat. 352360, Franklin Lakes, NJ, USA). We aspirated the solution and resuspended the pellet in 1 mL of Dispase II (Gibco, 1U/mL in PBS, Shanghai, China) to break up tissues. The solution was transferred into a 1.5-mL tube and incubated at 37 °C for 20 min. Then 1 mL of washing buffer (Hanks buffered saline solution containing 20% FBS, 5 mM CaCl₂, and 0.5 u/mL DNAse) was added to the samples. The samples were treated with 0.2 mL of PBT (100 µg/mL RNase A and 50 00B5g/mL PI in PBS). Setting 200 µL PBS as the control group, we commenced dark treatment at room temperature for 30 min. We resuspended the cells in 1 mL PBS (with 2–3% FBS), which was then passed through a 40-µm nylon filter (BD Falcon, Cat. 352340, Franklin Lakes, NJ, USA), and then we counted the cells. The labeled cells were detected for PI staining using a Beckman Coulter CytoFlex 5, and the percentages of cells in the G0/G1, S, and G2/M phases in each sample were determined via gating using the Flowjo 10.0 software (Beckman Coulter, Brea, CA, USA).

FRα expression in the panel of OC cell lines was determined using flow cytometry. Briefly, the chosen panel of OC cell lines as well as the breast cancer cell line, MCF7 (used as a negative control) were plated onto 24-well plates overnight at a seeding density of 1.24 × 10⁵ cells per well^{53 (link)}. Cells were left to adhere for 24 hours before being scraped and collected. Treatment groups were incubated with an anti-FRα primary antibody (1:100, ab3361, Abcam, Toronto, ON, Canada) while control group cells were incubated with PBS at 4 °C for 30 min. Cells were then pelleted via centrifugation, washed twice with ice cold PBS before incubation with the Alexa Fluor® 488-labelled secondary antibody (1:2000, ab150113, Abcam, Toronto, ON, Canada) for another 30 min. After treatment with the secondary antibody, cells were pelleted and washed twice before resuspension in 500µL of cold PBS for analysis using flow cytometry (CytoFLEX 5, Beckman Coulter, Mississauga, ON, Canada) wherein mean fluorescence intensity of the gated cells (10,000 events) were collected and normalized to that of untreated cells (mean FITC treated divided by mean FITC untreated).