Vacutainer glass tube

Serum samples were obtained from enrolled subjects and processed within 2 hours. Peripheral whole blood samples were collected in 5 ml BD Vacutainer glass tubes without additive (BD Biosciences) and allowed to clot at room temperature for 30 min. Serum was separated by centrifugation at 3000 rpm for 15 min at 4°C. The samples were then divided into aliquots and stored at −80°C.

Seed culture was grown in 20 mL of BG-11 with 50 mM NaHCO₃ and appropriate antibiotics. The strains were grown under 50 µE/s/m² light condition with continuous shaking at 30 °C. The cyanobacterial culture was fed with 50 mM NaHCO₃ (add 1 mL of 1 M NaHCO₃ dissolved in BG-11) every day until OD730 reached 2–3. Then the culture was diluted to OD730 of 0.5, and grown in 5 mL of BG-11 medium with 50 mM NaHCO₃, appropriate antibiotics, 40 µM d-pantothenic acid (hemicalcium salt), 0.2 mM thiamine pyrophosphate, and 0.5 mM IPTG. The culture was grown under 50 µE/s/m² light intensity in a BD vacutainer glass tube at 30 °C. A low oxygen condition was created by flushing the tube headspace with nitrogen once per day in order to decrease acetyl-CoA consumption by endogenous TCA cycle. The cyanobacterial culture was fed everyday with 50 mM bicarbonate (add 250 μL of 1 M NaHCO₃ dissolved in BG-11). The growth was monitored by a Beckman Coulter DU800 spectrophotometer at 730 nm.

Peripheral venous blood samples were collected from fasting patients or healthy donors with written informed consent from each patient or healthy donor according to the institutional review board (IRB) protocols at UCLA and Cedars-Sinai Medical Center. Each 8.0 mL blood sample was collected in a BD Vacutainer glass tube (BD Medical, Fisher Cat. #02-684-26) with acid citrate dextrose. Samples were processed according to the manufacturer’s protocol within 4 h of collection. The final plasma samples were collected for the HCC EV study after centrifugation at 10,000 × g for 10 min. The plasma samples were aliquoted and stored in −80 °C refrigerators. Five hundred microliter plasma samples were then incubated with TCO-conjugated anti-EpCAM (250 ng), anti-ASGPR1 (125 ng) and anti-CD147 (125 ng) at room temperature for 30 min before being loaded into the EV Click Chips for the HCC EV purification. All plasma samples subjected to EV Click Chips and downstream RT-ddPCR assay underwent only one freeze-thaw cycle.

Overnight culture was inoculated (2% vol/vol) in fresh LB medium and grown to the mid-log phase. IPTG (0.2 mM) and 20 mM xylose were added into culture for protein synthesis at 30 °C for 6 h. Ten milliliters of culture were harvested and re-suspended into 2 mL fresh LB medium supplemented with 0.1 mM IPTG, 30 mM xylose, and C1 compounds (50 mM formate or 200 mM methanol) in a glass tube (BD vacutainer glass tube). Supernatant of culture was diluted for five-fold and filtered by Amicon 10 kDa protein filters. Twenty microliters of sample were applied to HPLC (high-performance liquid chromatography) with a Bio-Rad Aminex HPX87 column (30 mM H₂SO₄; 0.4 mL/min; column temperature 30 °C). Organic acids were detected using a multiple wavelength detector at 210 nm, and xylose and methanol were measured by a refractive index detector. Ethanol was determined by GC-FID (flame ionization detector) (Agilent Technologies). 1-Propanol was used as the internal standard.