Centrifuge 5702

Cells incubated with various agents for different times depending on the task were separated from the cultural flasks as described above. Next, the cells were transferred to test tubes, where complete medium was added in the ratio of cell suspension: complete medium = 1:3, centrifuged in an Eppendorf 5702 centrifuge for 5 min, 600 rpm, and the precipitate was dissolved in 1 mL of saline solution. Samples were analyzed on a CyFlow flow cytometer, Sysmex-Partec. For each sample, 50,000 events were recorded. To detect the fluorescence of the analyzed samples, a laser excitation at 488 nm was used collecting emission above 530 nm.

Graphite
powder (Sigma-Aldrich, 325 mesh) was used as the source material to
produce graphene flakes. The powder was used as received and dispersed
into a liquid medium consisting of EtOH and CAB. In detail, CAB (10
mg mL^–1) was dissolved in a closed beaker containing
EtOH on a hot plate under magnetic stirring set to 65 °C until
a clear solution was obtained. The graphite powder (40 mg mL^–1) was added and mixed briefly to obtain a homogeneous dark/black
mixture. This graphite mixture was transferred in 20 mL aliquots to
Falcon centrifuge tubes for the next sonication step. The exfoliation
took place in a bath sonicator (Branson 2510, ∼40 W output)
for 2- and 4 h intervals to evaluate the influence of the sonication
time on the final exfoliation effectiveness as well as the impact
on the material’s use in percolative conductive films. Following
sonication, the mixtures were centrifuged in three 30 min steps: at
500, 2000, and 4400 rpm using an Eppendorf 5702 centrifuge. After
the first two steps, the sediment was collected and set aside, while
the supernatant underwent the final centrifugation stage. After the
final centrifugation, the sediment was washed three times in pure
EtOH at 4400 rpm to remove the excess CAB. The final graphene dispersions
were obtained by redispersing the washed flakes in 5 mL of EtOH via
brief sonication.

Exo-metabolome (conditioned media) samples were lyophilized ~48 hrs using a VirTis BenchTop 4 K Freeze Dryer. Dried material was directly extracted in 10 mL methanol in 20 mL glass vials stirred overnight. Vials were centrifuged at 2750 × g for 5 min in an Eppendorf 5702 Centrifuge using rotor F-35-30-17. The resulting supernatant was transferred to a clean 20 mL glass vial and concentrated to dryness in an SC250EXP Speedvac Concentrator coupled to an RVT5105 Refrigerated Vapor Trap (Thermo Scientific). The resulting powder was suspended in methanol and analyzed directly by HPLC-MS, as described below. Endo-metabolome (nematode bodies) were lyophilized for 18-24 hrs using a VirTis BenchTop 4 K Freeze Dryer. Dried pellets were transferred to 1.5 mL microfuge tubes and disrupted in a Spex 1600 MiniG tissue grinder after the addition of two stainless steel grinding balls to each sample. Microfuge tubes were placed in a Cryoblock (Model 1660) cooled in liquid nitrogen, and samples were disrupted at 1100 RPM for 60 s. This process was repeated two additional rounds for a total of three disruptions. Pellets were transferred to 8 mL glass vials in 5 mL methanol and stirred overnight. Subsequent steps for concentration and resuspension were followed as described for the exo-metablome.

Approximately 500 000 cells of the cell‐line A549 were seeded in cell culture flasks (25 cm²) and grown in DMEM (10 % FCS, 0.2 % P/S, 0.9 % GM). After 24 h, the supernatant media was removed and the nonconfluent cell monolayer was reloaded with substance‐loaded medium (or a blank fresh medium as a control). After 24–72 h, the supernatant medium was collected and the cell monolayer was washed with PBS. The combined media and PBS were centrifuged (1500 rpm, 5 min, Eppendorf 5702 centrifuge). The pellet of dead cells was gently suspended in PBS (1 mL) and centrifuged again (1500 rpm, 5 min, 278 K, Eppendorf Centrifuge 5418). PBS was removed and lysis buffer (30 μL, 0 °C, 10 min) was added. Then RNAse (100 μg mL⁻¹, 10 μL) was added and the cells were incubated for 10 min on ice followed by a prolonged incubation at 37 °C for 2 h. To finally digest the cell proteins, the cell pellet was treated with protein kinase K (10 μL) at 52 °C for 12 h. The extract was mixed with DNA‐ladder dye (10 μL) and analyzed by gel electrophoresis (2 % agarose loaded with 10 μL ethidium bromide (10 mg mL⁻¹), 150 mV, 2 h, TAE buffer). The DNA bands were analyzed (see Figure S9 in the Supporting Information) by using a UV transilluminator (BioRad).

Exo-metabolome (conditioned media) samples were lyophilized for 24 h using a VirTis BenchTop 4 K Freeze Dryer. Dried material was directly extracted in 3 mL methanol with gentle rocking at room temperature. Following overnight extraction, the samples were centrifuged (2,750 × g, 22 °C, 5 min) in an Eppendorf 5702 Centrifuge. The supernatant was transferred to a clean 8 mL glass vial and concentrated to dryness in an SC250EXP Speedvac Concentrator coupled to an RVT5105 Refrigerated Vapor Trap (Thermo Scientific). The powder was suspended in 150 µL methanol, vortexed vigorously for 30 s, and sonicated for 5 min. The suspension was transferred to a 1.7-mL Eppendorf tube and centrifuged (18,000 × g, 22 °C, 5 min), and the clarified supernatant was transferred to HPLC vials and analyzed directly by HPLC-HRMS, see below.

The CNT suspension was prepared by dispersing 0.005 g of CNT powder containing a mix of single-wall and multiwall nanotubes (Aldrich Chemistry, St. Louis, MO, USA) into 10-mL of isopropyl alcohol (IPA). The suspension was then centrifuged in an Eppendorf 5702 Centrifuge (Eppendorf AG, Hamburg, Germany) at 3000 rpm for 15 min. A pipette (Labnet, Edison, NJ, USA) was used to collect the supernatant with homogeneously distributed CNTs.

Blood samples were collected by venipuncture into 10 mL draw capacity BD Vacutainer tubes containing 18 mg of K₂EDTA (366643, BD Vacutainer EDTA, Becton, Dickinson and Company, Franklin Lakes, NJ). Participants were asked to provide fasting blood donations, and most did (Supplementary Fig. 2B). Following collection, tubes were stored upright at room temperature for at least 30 min until centrifugation within 1 h of collection and processing to obtain PFP. Briefly, blood was centrifuged at room temperature (22–25 °C) for 10 min at 450×g in an Allegra 6 centrifuge (Beckman Coulter, Inc., Pasadena, CA). The plasma layer was removed without disturbing the interface and pipetted into a fresh 15 mL Falcon tube. The plasma was then centrifuged for 15 min at 2500×g in an Eppendorf 5702 centrifuge (Eppendorf, Hamburg, Germany) and the supernatant transferred to a new 15 mL Falcon tube. This centrifugation process was repeated once more. The resulting PFP was transferred to 1.5 mL Eppendorf tubes in 500 μL aliquots, the aliquots were flash frozen on dry ice and stored at − 80 °C until RNA isolation.