Slgv033rs

Five F. tularensis strains of subsp. tularensis Nevada 14 and subsp. holarctica LVS, Kato, Yama, and Kf Water were prepared to validate the effective inactivation of F. tularensis. Bacteria were heated at 94°C for 3 min and 56°C for 30 min, filtered using Millipore PVDF Hydrophilic Millex-HV Sterile Syringe Filter Unit 0.45 Micron (SLHV033RS, EMD Millipore Corporation) and 0.22 Micron (SLGV033RS, EMD Millipore Corporation), incubated at 4°C for 24 h with detergents (1% LDS buffer, 1% NP-40, and 1% TritonX-100), and radiated at room temperature (23°C) with a low-pressure lamp (254 nm). These procedures were in accordance with the same conditions as those for the treatment of F. tularensis SCHU P9. The CFU of untreated and treated samples were compared. The experiments were conducted in four replicates.

Fifty microliters of F. tularensis SCHU P9 (average, 2.9 × 10⁶ CFU/ 100 μL) suspended in 1 mL of CDM was screened through Millipore PVDF Hydrophilic Millex-HV Sterile Syringe Filter Unit 0.45 Micron (SLHV033RS, EMD Millipore Corporation, Billerica, MA, USA) and 0.22 Micron (SLGV033RS, EMD Millipore Corporation). Before and after filtration, the samples were serially diluted and cultured on Eugon chocolate agar plates at 37°C for 4–7 days. The average CFU number in 100 μL was calculated from the average number of colonies of four replicated samples.

Cell media were prepared with DNAse- and RNAse-free water and filtered through 0.22 μm filter membranes (Millex GV, SLGV033RS, Millipore, Billerica, USA) prior to use. Cell cultures were manipulated using sterile, disposable nonpyrogenic plastic ware and were maintained at 37°C in an atmosphere of 5% CO₂ in air at a relative humidity of 80%. Human breast epithelial cells MCF-10A (ATCC) were cultured in a 1 : 1 (v/v) mixture of Dulbecco's Modified Eagle's Medium (DMEM, 12100046, Gibco, Waltham, USA) and Ham's F12 nutrient mixture (HamF12, 21700-075, Gibco, Waltham, USA) supplemented with 5% inactivated horse serum (16050-130, Gibco, Waltham, USA) 10 μg/mL insulin (PHC9624, Gibco, Waltham, USA), 0.020 μg/mL human epidermal growth factor (EGF, PHG0311, Gibco, Waltham, USA), 0.5 μg/mL hydrocortisone (H0888, Sigma Aldrich, St. Louis, USA), 0.10 μg/mL choleric toxin (C8052, Sigma Aldrich, St. Louis, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin (P4333, Sigma Aldrich, St. Louis, USA). Cells were routinely trypsinized and inoculated onto plates at a density of 4 × 10⁴ cells/cm². Every month, cells were cultivated in the absence of antibiotics for control purposes and subjected to routine assay using a MycoAlert Mycoplasma Detection kit (LT07, Lonza, Walkersville, USA).

Uranyl formate (0.75%) solution was freshly prepared by adding 5 mL boiling water to 37.5 mg uranyl formate (Electron Microscopy sciences, 22450). After stirring for 5 min in the dark, 10 μL 5 M NaOH was added and stirred for an additional 5 min. The solution was syringe filtered (Millipore, SLGV033RS) and stored in the dark. Purified Gpr126 (−ss), (+ss), and (−ss) D134A/F135A ECR constructs were diluted to ~5 ug mL⁻¹ and applied to glow-discharged EM grids (Electron Microscopy Sciences, CF400-Cu,) using a conventional negative-stain protocol⁸⁶ . To the grid was applied 2 μL diluted protein for 30 sec. The protein was blotted off with filter paper (Sigma–Aldrich, WHA1001110), and then the grid was touched to a 25 μL drop of distilled, filtered water. The water was blotted off, and the grid was touched to a second 25 μL drop of water and blotted off. The grid was then touched to a 25 μL drop of 0.75% uranyl formate for 30 sec and blotted off. The grid was air-dried for 30 sec. The sample was imaged on a Tecnai G2 F30 operated at 300 kV. Gpr126 −ss (6565 particles), +ss (2529 particles), and −ss D134A/F135A (3916 particles) were processed using EMAN2^{87 (link)}.

Vegetative cells were treated with 100 µg/mL cycloheximide for 5 min prior to harvesting. Cells were pelleted by centrifugation and resuspended in buffer KK₂ (16.5 mM KH₂PO₄, 3.9 mM K₂HPO₄, 2 mM MgSO₄) plus 100 µg/mL cycloheximide. They were washed twice more in KK2, with a final wash in KK₂ containing 100 µg/mL cycloheximide and 1× SigmaFast EDTA-free protease inhibitor cocktail (Sigma #S8830). The cell pellet was resuspended at 2 × 10⁸/mL in 50 mM HEPES pH 7.5, 40 mM Mg(CH₃COO)₂, 25 mM KCl, 5% sucrose, 0.4% IGEPAL® CA-630 (Sigma #I8896), 100 μg/mL cycloheximide, 1× SigmaFast EDTA-free protease inhibitor cocktail, 2 mM PMSF and lysed by passing through a 25 mm diameter Swin-Lok filter holder (GE Healthcare Life Sciences #420200) containing a prefilter (Millipore #AP1002500) together with a 5 µm nucleopore track-etched membrane (Whatman #110613). The lysate was cleared by centrifugation (8,000 g for 30 min at 4 °C) and the supernatant passed through a 33 mm Millex-® GV 0.22 µm PVDF filter unit (Millipore #SLGV033RS). The filtrate was divided into 1.4 mL aliquots after A₂₆₀ determination, flash-frozen in liquid N₂, and stored at −80 °C. All buffers were at 4 °C.

Exosomes were isolated from the conditioned medium of ADMSCs cultured under 2D, 2.5D, and 3D conditions by standard ultracentrifugation (UC) methods with slight modifications, referring to Ref. [44 (link)]. Briefly, to eliminate fragments and dead cells, we first pelleted intact cells at 300 × g for 10 min, then dead cells at 2000 × g for 30 min. Cell- and fragment-free conditioned medium was purified by pelleting at 10 000 × g for 30 min. The supernatant was filtered through a 0.22-μm filter (SLGV033RS, Millipore, USA) to remove vesicles or protein aggregates. Then the supernatant was spun at 100 000 × g for 70 min. The resulting pellet was washed with PBS, transferred to a new tube, and spun at 100 000 × g for 70 min. The pellet was resuspended for subsequent experiments. All spins were performed using an Optima MAX-XP ultracentrifuge (Beckman Coulter, USA) at 4°C.