Microfuge 22r

Cells were seeded in 6‐well plates, and the activity of autophagic sequestration was measured as the transfer of the cytosolic enzyme LDH to sedimentable autophagic vacuoles, in the presence of 200 nM BafA1 (Enzo, BML‐CM110) with or without starvation (in EBSS medium) for 3 h as previously described 45. Instead of using the high‐density cushion for separating soluble from the sedimentable LDH, the disruptate was diluted 2.5 times with resuspension buffer (50 mM sodium phosphate, 1 mM EDTA, 1 mM DTT) containing 0.5% BSA and 0.01% Tween‐20 and sedimented it for 50 min at 20,000 × g (Beckman Coulter Microfuge 22R). Subsequent steps in processing and LDH measurement were as previously described. After accounting for the dilution factors and calculating the ratio of sedimentable over soluble LDH, the final values presented are net sequestered LDH per hour (%/h), where the background value has been subtracted (LDH amount found in untreated/control samples) and incubation time accounted for 36, 45.

For in vitro platinum treatment, extracted naked genomic yeast DNA in TE was incubated with cisplatin or oxaliplatin, added from stock solution giving the required final drug concentration. DNA was incubated with cisplatin or oxaliplatin for 2 hours at 37°C and DNA recovered by ethanol precipitation. Precipitation was conducted by adding 2.5 × volume 100% ethanol, incubating at -80°C for 20 minutes, then at −20°C for 30 minutes, followed by centrifugation at 13500 rpm (Beckman-Coulter Microfuge 22R) for 20 minutes at 4°C. DNA pellets were washed with 75% ethanol and centrifuged a second time at 13500 rpm for 20 minutes at 4°C. Pellets were dried using a SpeedVac system (Thermo Savant) and resuspended in 100 μl TE prior to sonication and DIP.

After 20 min, PRF clots were retrieved from the vacutainers and RBC layer was detached and discarded. Four PRF clots were transferred into sterile tubes and were agitated gently for 5 min (Agitaser^®, Barcelona, Spain). The clot was then minced in a 7 mL tissue grinder (Tenbroeck^®, Bengaluru, India) to obtain a releasate which was measured and the releasate returned into the tube. The releasates were immediately centrifuged at 10,000 g for 15 min (Microfuge22R^®, Beckman Coulter, Fullerton, CA) to pellet out any residual blood cells, and supernatants were frozen at −80°C till determination of the growth factors (VEGF and epidermal growth factor [EGF]). Two commercially available ELISA kits were used to measure VEGF[8 (link)] (PicoKine™, Bosterbio, Pleasanton, USA) and EGF[9 ] (Human EGF ELISA Kit^®, Origene, Rockville, USA) levels, respectively, as per the instructions of the manufacturers.[8 (link)9 ]

Blood (0.9 ml) was added to 0.1 ml of filtered Caltag A and allowed to stand at room temperature for approximately 60 minutes. Red and white cells were removed by a 20-minute spin (20°C) at 300g using a swinging bucket centrifuge with brake set at 0 (Allegra X-22 R; Beckman Coulter, Pasadina, CA). The supernatant (0.4 ml; platelet-rich plasma) was removed with a wide-mouth pipette tip (2 × 200 μl) and mixed thoroughly with 0.6 ml of minimal buffer citrate (standard NaCl and KCl, with 10 mM Hepes and 11 mM sodium citrate). This was further spun at 2500g for 20 minutes (same centrifuge) to produce a platelet pellet and platelet-free supernatant; 0.8 ml of the latter was then removed, 200 μl saved, and the remaining 600 μl spun at 15,000g for 30 minutes at 20°C (swinging bucket, Microfuge 22R; Beckman Coulter). Four hundred microliters of supernatant was removed and half of this was saved (high-speed supernatant), leaving 200 μl above the high-speed pellet (HSP). All samples thus consisted of 200 μl of sample, and each of these was diluted with an equal volume of filtered minimal buffer and 10% DMSO and frozen (5 minutes at − 27°C, plunged to dry ice or at a lower temperature, and stored at − 75°C). The vast majority of MVs was contained in the HSP, and this is the basis of the data presented.

SERS measurements were performed on a customized Raman microscope equipped with Acton SP-2500i spectrograph (Princeton Instrument, US) and Pixis-100BR CCD (Princeton Instrument, US) in the whole experiment. A He–Ne laser (14 mW) was used in the experiments. Laser power 12 × mW, 50× objective, and 5 s integration time were used in the experiments. At least 10 spots on the same G-SERS sensor were examined. The Ag NPs was centrifuged in a centrifuge (Microfuge® 22 R, Beckman coulter, US) at 10,000 rpm for 10 min at 20°C. The Ag NPs were characterized by a Field Emission Transmission Electron Microscope (TEM, JEM-2100 F, Japan), and the gum and G-SERS sensor by a scanning electron microscope (SEM, FEI Inspect F), respectively. The sampling recovery efficiency was determined by a UV–Vis spectrophotometer (UV–Vis, UV-4802H, China) at the wavelength of 614 nm. Liquid chromatography-mass spectrometry (LC-MS) measurements were performed on Shimadzu LCMS-8045 (USA).