Microscope slide

The produced W/O/W emulsions and centrifuged W/O/W were added onto glass slides (VWR Microscope Slides with cut edges, 1.0 mm thickness, 76 × 26 mm). W/O/W emulsions and centrifuged W/O/W emulsions were imaged using confocal microscopy (Leica TCS SP8, Leica Microsystems Inc. and Leica TCS SP8 STED 3×, Leica Microsystems Inc.). The crowding sensor crGE2.3 was excited at 488 nm and the emission was split into a 510–525 nm channel for the donor detection and a 600–700 nm channel for the FRET detection. For the acceptor excitation a wavelength of 561 nm was used and a 600–700 nm channel was used for emission detection. DiD was excited at a wavelength of 633 nm and for emission a 650–720 nm channel was used. All data were analyzed using ImageJ.

For all crowding experiments, except for the crowding experiments with the crowding sensor (crGE2.3), were done on glass slides (VWR Microscope Slides with cut edges, 1.0 mm thickness, 76 × 26 mm). The W/O/W emulsions, in‐flow emulsions, or vesicles (20 µL) were added onto a glass slide and immediately after the NaCl(aq.) solution was added. After 5 min the crowding was observed and characterized. For the W/O/W crowding experiments with crowding sensor (crGE2.3), an Eppendorf (1.5 mL; Eppendorf Tubes) was filled with the aqueous NaCl solution (100 µL) with double the concentration of the targeted final concentration. The W/O/W emulsion (100 µL) was added and after 5 min the crowded W/O/W emulsion could be characterized. The procedure for the centrifuged WO/W crowding experiments with crowding sensor was identical, except that for both salt solution and W/O/W emulsion 300 µL instead of 100 µL, respectively, was used. After 5 min, the crowded W/O/W emulsion was centrifuged (14 100 rcf, 15 s) and could be collected from the bottom of the Eppendorf tube.

4 × 10³ cells/cm², either attained directly from culture flasks or derived from fresh 3^rd generation disaggregated spheres (in the case of SC-DRenG2 and SC-DDRenG2 cells), were seeded on the top of microscope slides (VWR) placed inside a 100 mm cell culture dish (SPL-Biosciences®) and cells were allowed to grow until approximately 80% confluence. Following medium aspiration the slides were rinsed twice with PBS (Sigma-Aldrich®), collected into centrifuge tubes (SPL-Biosciences®) containing 50 mL of 95% ethanol (Sigma-Aldrich®) and kept overnight at 4 °C. To quench the endogenous peroxidase activity 15 min incubation was performed in a 3% hydrogen peroxide (H₂O₂) solution. Subsequent preparation steps were performed using the Ultra Vision Kit (Thermo Scientific®) according to manufacturers’ instructions. After dehydration, slides were mounted using the Tissue-Tek Glas Mounting Medium (1408, Sakura).
Vimentin was stained using the Vim3B4 primary antibody (Dako Corporation), α-smooth muscle actin the αSM-1 (Leica Biosystems), OCT3/4 with the N1NK (Leica Biosystems) and β-catenin with CAT-5H10 (ThermoFisher Scientific). DAPI staining was used to identify the nuclei. Cells’ were observed in a Nikon Eclipse 80i microscope and photographs were taken using a Nikon Digital DXM1200F coupled camera.

Anhydrous dimethylsulfoxide (DMSO) (99.9%, Product 276855), anhydrous dimethylformamide (DMF) (99.8%, Product 227056), anhydrous tetrahydrofuran (THF) (99.9%, inhibitor-free, Product 401757), anhydrous acetonitrile (ACN) (99.8%, Product 271004), 10K polyethylene glycol (10K PEG) (BioUltra, Product 92897-F, Lot BCCB7356), (1,4-diazabicyclo[2.2.2]octane) (DABCO) (99%, D27802, Lot WXBC2274V), and 1,6-diisocyanatohexane (HMI) (99%, Product 52649, Lot BCBZ8006) were purchased from Sigma-Aldrich. di-n-butyltin dilaurate (DBTDL) (>95%, Product 71130, Lot W24B0211H), 1H,2H,2H-perfluorooctyltrichlorosilane (FOTS) (96%, Product L16606, Lot 101218683) was purchased from Alfa Aesar. 1,1,1-tris(hydroxymethyl)ethane (TME) (97%, Product 824324, lot R27C029) and microscope slides (Product 631-1553) were purchased from VWR. Western Blot Mini-Protean slides were purchased from BioRad (Product 1653308). Silicone gaskets were fashioned from high-temperature silicone rubber sheets from MacMaster-Carr (Products 3788T21, 3788T22).

In addition to stock solutions, the self-driving laboratory uses consumable glass substrates (75 mm × 25 mm × 1 mm microscope slides; VWR catalog no. 16004-430), 2 mL HPLC vials (Canadian Life Science), and 200 µL pipettes (Biotix, M-0200-BC). These are placed in appropriate racks and trays for access by the robotics.
The HPLC vials and pipettes were used as received, whereas the microscope slides were cleaned by sequential sonication in detergent, deionized water, acetone, and isopropanol for 10 min each^{8 (link)}. Wells of 18 mm diameter were then created on the microscope slides using a sprayed enamel coating (DEM-KOTE enamel finish) and circular masks placed at the center of each slide (Supplementary Fig. 1). The wells serve to confine the precursor solution before it dries.