Tryplexpress

In vitro emboli formation was carried out as described (23 (link)). Briefly, cells were trypsinized 24 hours after nucleofection (if applicable); 100,000 cells were seeded in 6-well ultra-low attachment plates (Corning, 3471) in medium containing 2.25% PEG8000 and gently rocked at approximately 40 rpm for 3–4 days or as indicated. To isolate emboli, cultures were centrifuged at 27g for 1 minute with PBS, treated with TrypLExpress (Thermo Fisher Scientific, 12604-013) for 5–10 minutes and neutralized with cell culture medium. Cells were counted with a Countess (Thermo Fisher Scientific) using trypan blue dye exclusion. Unless indicated otherwise, all analyses of emboli were conducted after 4 days in 3D culture. For assessment of cell death within established emboli, these were generated first by seeding 10,000 cells per well in 96-well plates (Nexcelom Biosciences, ULA-96U-010), cultured and treated as indicated, followed by addition of PI (0.5 μg/mL) for 30 minutes and imaging with an EVOS FL microscope. For quantification, emboli were harvested as above; cells were transferred at 10,000 cells/well in 96-well plates. Six hours later, they were treated with PI for 30 minutes and analyzed by Direct Cell Counting (Celigo, Nexcelom).

Colon crypts from 4- to 7-month-old mice were isolated as described above and then resuspended in 4 ml of TryplExpress (ThermoFisher Scientific) for 15 min at 37 °C under agitation at 75 rpm. Ups-and-downs were performed with a P1000 pipette and incubated for a further 25 min at 37 °C and 75 rpm. After a second round of ups-and-downs, 8 ml of binding buffer [PBS-2 mM EDTA-2% BSA (Sigma-Aldrich) (w/v)] was added and the mix was passed through a 40 µm filter (Avantor). Cells were centrifuged at 1300 rpm for 10 min and the pellet was washed with 4 ml of binding buffer. After centrifugation at 1300 rpm for 3 min, cells were incubated with a Phycoerythrin-coupled anti-Epcam antibody (BD Biosciences) at 1/100 (v:v) in binding buffer for 30 min on ice. After 3 washes with binding buffer, cells were sorted using a FACS Aria I cytometer (BD Biosciences). FSC and SSC intensities were used for debris and doublets’ exclusion. GFP^+ve cells were identified using the FITC channel (Olfr78-GFP line) and Epcam through the PE channel. Epcam^+ve/GFP^+ve cells were sorted from 2 pools of 2 HE (1028 and 2511 cells for each pool) and 2 KO mice (1123 and 1506 cells for each pool). Five thousand Epcam^+ve/GFP^-ve cells were collected from a WT mouse as 2 individual samples. The cells were collected in QIAzol lysis reagent (Qiagen).

We used human GBM cell lines that were established from tumor samples from two patients and named the cell lines no. 1 and no. 2. To establish the cells, we minced and pipetted the tumor samples in TrypL Express (Thermo Fisher Scientific, Waltham, MA, USA) and incubated them in a CO₂ incubator at 37 °C, cells were rinsed and collected after filtering through a 40-µm cell strainer (Thermo Fisher Scientific) to remove any aggregates. Use of human materials and protocols were approved by the Ethics Committees of Kanazawa University and Kyoto University (R0534). Cells were cultured as non-adherent spheroids in serum-free DMEM/F12 containing GlutaMax (Thermo Fisher Scientific), B27 without vitamin A (Thermo Fisher Scientific), penicillin and streptomycin (Nacalai Tesque, Kyoto, Japan), hEGF (20 ng/mL) and hFGF (20 ng/mL, Peprotech, TX, USA). To induce cell differentiation, we exposed the cells to media containing 10% FBS for 24 h. We dissociated cells to single cells using accutase (Nacalai Tesque) and rinsed them with PBS before mass cytometry analysis.

Gonads were collected from male pouch young (n = 17; Supplementary Data 5) and germ cells were isolated from the gonads as previously described^{88 (link)}. Briefly, gonads were torn using needles in a droplet of TrypLExpress (Gibco, 12605010) and incubated at 37 °C for 20 min. Tissue clumps were triturated manually by gentle strokes with a P1000 pipette tip and then with a P200 pipette tip. TrypLExpress was diluted with 5% FBS (in PBS), filtered through a 40 µm cell strainer, and washed twice in PBS (centrifugation 240 × g for 2 min). The resulting pellet was resuspended in Zombie Near-Infra Red Viability Dye (BioLegend, 423105) and incubated at room temperature for 20 min. The lysate was washed in 5% FBS (in PBS), then resuspended in SSEA1 monoclonal antibody (Invitrogen, MC-480) conjugated to DyLight 488 (1:300 in 5% FBS (in PBS); Invitrogen, MA1-022-D488) on ice for 30 min. The lysate was then washed, resuspended in sort buffer (5% FBS, 0.5 mM EDTA in PBS), and kept on ice prior to cell sorting. Validation of the SSEA1 monoclonal antibody for germ cell specificity in brushtail possum was previously undertaken^{74 (link)}.

Human breast cancer cells MCF-7 (ATCC HTB-22) were used for the in vitro studies. The cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) media with 10% heat inactivated Fetal Bovine Serum (FBS) (FBS; Gibco 306.00301). The medium was supplemented with1% penicillin/streptomycin (PAA Laboratories GmbH, P11-010) and 1 µg/mL Amphotericin B (PAA Laboratories GmbH, P11-001).
Human skin fibroblast monolayer cultures (WS1-ATCC CRL1502) were grown in Eagle’s minimal essential medium (Invitrogen 32360–026) that was modified to contain 2 mM L-glutamine (Gibco, 25030), 1 mM sodium pyruvate (Gibco, 11360), 0.1 mM nonessential amino acids (Gibco, 11140), 1% amphotericin-B (Gibco, 104813), 1% penicillin–streptomycin (Gibco, 15140) and 10% v/v foetal bovine serum (FBS; Gibco, 306.00301).
Cells were maintained in a CO₂ incubator (37 °C, 5% CO₂ and 80% humidity). Cells were washed with Hank’s Balanced Salt Solution (HBSS, Invitrogen, 10–543 F) when it became confluent and detached with TryplExpress (Gibco, 12604) and subcultured. Cells were seeded at a concentration of 5 × 10⁵ cells/plate in 3.5 cm² diameter culture plate.

Human breast adenocarcinoma cells (MCF-7, ATCC HTB-22) were used in this study. Dulbecco’s modified Eagle medium (DMEM) was used for cell culture with 10% fetal bovine serum (FBS; Gibco 306.00301), 1% penicillin/streptomycin (PAA Laboratories GmbH, P11-010), and 1 µg/mL amphotericin B (PAA Laboratories GmbH, P11-001). Cells were grown at 37 °C, 5% CO_2, and 80% humidity in a CO₂ incubator. Hank’s Balanced Salt Solution (HBSS, Merck, Johannesburg, South Africa) was used to wash cells when confluent. Cells were detached using 1 mL/cm² TryplExpress (Gibco, ThermoFischer Scientific, 12604, Johannesburg, South Africa) for subculturing. For experiments, 5 × 10⁵ cells were seeded in 3.5 cm diameter cell culture plates, allowed to attach for 6 h, and treated/incubated for 24 h with 10 µg/mL of RF, ZnO NPs, and RFZnO NPs for each experiment.