Falcon tube

Explants were generated by removing the entire colon or the distal 15 cm of the SI. Any remaining connective tissue was removed from the outside of the tissue and then tissue was gently flayed open. Fecal and mucus contents were gently removed with forceps and placed in a 6-well plate with 1× PBS. After a brief wash with 1× PBS, tissues were placed in 15 mL Falcon tubes (VWR) in a tissue culture hood with 5 mL of complete RPMI 1640 medium (10% FBS, HEPES, sodium pyruvate, nonessential amino acids, 100 U/mL penicillin/streptomycin, and l-glutamate). Tubes were placed horizontally in a container and shaken for 25 minutes at room temperature (RT), at a speed sufficient to maintain gentle movement of the tissue. After this incubation, tissues were plated in 10 cm dishes with 20 mL of complete exosome-depleted RPMI (the same media as complete RPMI above but supplemented with 10% exosome-depleted FBS, Systems Biosciences) and then placed in a 37°C 5% CO₂ incubator for 16 hours. The supernatant of these plates was used to isolate vesicles, as described below.

Spiked blood samples were centrifuged at 2500g for 15 min and the layer with leukocytes and tumor cells was transferred to 15 mL conical Falcon tubes (VWR Bie & Berntsen, Soeborg, Denmark). Remaining red blood cells were lysed with FACS lysing solution (BD Biosciences) and the samples centrifuged at 2500g for 15 min. Thereafter, cells were stained using CTC Stain^™ comprising a mixture of anti-CD45/Near InfraRed (NIR) antibody (clone: HI30), anti-pancytokeratin/Green antibody (clone: AE1/AE3), 4′,6-diamidino-2-phenylindole (DAPI), and a permeabilization buffer (Intrastain, DAKO, Glostrup Denmark). Cells were then washed with phosphate buffered saline (PBS) with 1 % BSA and resuspended in H₂O, transferred to a CytoDisc^™, air-dried, and mounted using VectaShield Hard Set Mounting Medium (Vector Laboratories Inc., Burlingame, CA, USA) and covered with a CytoCover^™.

The following synthesis procedure was for CQD20, but all syntheses follow the same general format. 1.0 g citric acid and 2.0 g of urea were combined in 20 mL of Milli-Q water and sonicated for 10 min inside a 100 mL Teflon-lined autoclave were acquired from Shanghai Yuhua Instruments Equipment Co. Ltd, China. The steel autoclave used supports pressures up to 3 MPa. A VWR oven was set to warm up to and hold 160°C for 6 h, then cool down. After returning to room temperature, the autoclaves were removed from the oven and the solution was transferred directly into dialysis bags (Shanghai Yuanye Bio-Technology Co. Ltd, China) with a molecular weight cut-off (MWCO) of 1,000 Da. The solution was left to dialyze for at least 8 h, with the water being changed at least 6 times. The solutions were transferred to 50 mL Falcon tubes (VWR Canada), where a Kimwipe was attached to the top with an elastic band. This Falcon tube containing solution was frozen in liquid nitrogen and placed in a Labconco Lyophilizer at −84°C for at 48 h. The obtained product was light and fluffy and ranged from dark green to brown in color. These CQDs were stored in a refrigerator, sealed with Parafilm, and wrapped in tinfoil to prevent any degradation until use.

Salad was manually cut into small pieces before being weighed into falcon tubes (50 mL, VWR, Vienna, Austria) using a CPA225D balance (Sartorius, Vienna, Austria). Raspberries and strawberries (whole fruits) were homogenized with a hand blender (Tefal/SEB, Ecully, France). Raspberries, strawberries and deep-frozen homogenized spinach were directly weighed into 10 mL glass vials (more details can be found in Table A2). In order to prevent potential oxidation of lipids, 3 mL of an approximately 0.01% BHT solution in IPA were added and samples were mixed. Subsequently 30 µL IS were spiked into all samples except for one replicate (to test for potential IS presence in plants). Salad samples were homogenized using an ultra-turax (miccra d-1, Heitersheim, Germany) which was cleaned with 70% IPA and dried between the samples. In order to inhibit lipase activity, all samples were incubated at 75 °C for 30 min under constant shaking [27 (link)]. The warm salad samples were subsequently transferred into glass vials. The following sections provide a detailed overview of the extraction strategies that were applied.