On 6/12/17, 7/17/17, 8/8/17, 8/21/17, 9/11/17, 9/25/17, 10/30/17, and 5/7/18 the iron chloride procedure was used on the pond water after sequential 1 μm and 0.2 μm filtration, with the 0.2 μm a common pore size used in virome generation to prevent cellular contamination [33 (link), 37 (link), 42 (link)]. A 1 mL solution of FeCl3 (4.83 g FeCl3 into 100 ml H2O) was added to the filtered pond water and incubated in the dark for 1 h. The samples were then filtered onto 142-mm 1 μm polycarbonate filters (Sigma-Aldrich, MO, United States) to capture flocculated viral particles [43 (link)]. Filters were stored at 4 °C in the dark until resuspension. For resuspension, filters were rocked overnight at 4 °C in 10 mL of 0.1 M EDTA - 0.2 M MgCl2− 0.2 M Ascorbate Buffer, described in detail elsewhere [43 (link)]. Resuspended viral particles were then subjected to a DNase I (Sigma-Aldrich, MO, United States) treatment for 1 h and passed through a 33-mm diameter sterile syringe filter with a 0.2 μm pore size (Millipore Corporation, MA, United States). DNA was extracted from 500 μl of the viral concentrate using the AllPrep PowerViral DNA/RNA Kit (Qiagen, CA, United States) per the manufacturer’s instructions. Prior to sequencing, viral DNA was tested for the presence of bacterial contamination via 16S rRNA gene PCR.
Sterile syringe filter
Manufactured by Merck Group
Sourced in United States
The Sterile Syringe Filter is a laboratory equipment designed to remove particulates and microorganisms from liquids. It features a membrane that traps contaminants, ensuring the sterility and purity of the filtered solution.
Automatically generated - may contain errors
Lab products found in correlation
Bradford reagent, by Merck Group (3 mentions)
Fetal bovine serum (fbs), by Biowest (3 mentions)
Dulbecco modified eagle medium (dmem), by Biowest (2 mentions)
Exoquick exosome precipitation solution, by System Biosciences (2 mentions)
Allprep powerviral dna rna kit, by Qiagen (1 mentions)
Dnase 1, by Merck Group (1 mentions)
Optima l xp ultracentrifuge, by Beckman Coulter (1 mentions)
Centrifuge 5430 r, by Eppendorf (1 mentions)
Prestoblue cell viability reagent, by Thermo Fisher Scientific (1 mentions)
Synergy h1, by Agilent Technologies (1 mentions)
Mem alpha, by Biowest (1 mentions)
Dmem medium, by Thermo Fisher Scientific (1 mentions)
Sf 900 2 sfm, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle medium, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Tecnai g2 12, by Thermo Fisher Scientific (1 mentions)
16 protocols using sterile syringe filter
1
Viral Particle Concentration and Purification
2
Isolation of Extracellular Vesicles from Bacterial Culture
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The EVs were isolated using a previously described centrifugation and filtration protocol [20 (link)], with slight modifications. Briefly, for pelleting the bacteria, the broth culture was centrifuged at 5000 × g at room temperature for 10 minutes (Centrifuge 5430 R, Eppendorf AG, Germany). For removing any remnants of intact bacterial cells, the supernatant was filtered through a 0.22 μm sterile syringe filter (Millipore, Germany). The filtrate was then re-centrifuged at 125000 × g at 4° C for 3 hours (Optima™ L-XP ultracentrifuge, Beckman, USA). The obtained pellet was suspended in 300 μl sterile phosphate-buffered saline (PBS). The EVs samples were stored at -20° C until used.
3
Preparation and Characterization of AM-MSC Conditioned Media
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The CM were prepared as described in our previous publication (Humenik et al. 2019 [12 (link)]. Shortly, AM-MSCs (P2) were cultured in DMEM (Biowest) without FBS (Biowest). After 24 h incubation in a humidified atmosphere with 5% CO2 at 37 °C, collected media samples were filtered through a 0.2 µm sterile syringe filter (Millipore, Burlington, MA, USA). To ensure that equal concentrations (2.0 mg/mL) of CM were used for the subsequent experiments, the protein concentration of the CM was quantified by Bradford protein assay using standard Bradford reagent (Sigma). As a control (nonconditioned medium), DMEM was regarded. Samples of AMMSC-CM were collected and stored at −80 °C until use.
4
Phage Adsorption Assay for C. jejuni
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Phage adsorption assays were performed as previously described with minor modifications (Baldvinsson et al., 2014 (link)). Briefly, C. jejuni were grown overnight on BA plates and harvested in CBHI. Cells were pelleted by centrifugation at 6,000 × g for 5 min and re-suspended in CBHI. This washing procedure was repeated twice (total number of washes, three), and the final cell suspension was adjusted to an OD600 of 0.4. Bacteriophages were added to the bacterial suspension at a final concentration of 106 PFU/ml (multiplicity of infection [MOI] of approximately 0.0025) and incubated at 37°C with gentle shaking (50–80 rpm) for a total of 90 min. Samples containing free-phages were collected at 0, 15, 30, 60, and 90 min, filtered through an 0.22-μm sterile syringe filter (Millipore), and stored at 4°C until enumeration (plaque assay). Experiments were repeated twice and for each experiment, the calculated phage pfu/ml at time zero was designated as 100% free phages. The percentage of free phages was calculated for the remaining time points according to time zero. All experiments were performed in duplicate and the data represent the mean percentages of free phages and standard deviations thereof from the two independent experiments.
5
Cytotoxicity Evaluation of MOS and D-Mannose
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Cytotoxicity of the samples was assessed in the HeLa cell line according to ISO 10993–5 (2009 ), using PrestoBlue™ Cell Viability Reagent (Thermo Fisher Scientific, MA, USA) as per manufacturer’s instructions. The samples (MOS Parr, MOS H3PO4 and commercial D-mannose) were directly dissolved in DMEM medium and sterilized using a sterile syringe filter with a 0.22-µm pore size (Millipore, Billerica, MA, USA) at a concentration of 20 mg/mL. Decimal dilutions were performed to test the concentration range of 10.0–0.31 mg/mL. For the HeLa viability assay, suspended cells were placed into a 96-well microtiter plate at a seeding density of 1 × 104 cells/well. The cells were then cultured for a period of 24 h to create a semi-confluent monolayer. Following this incubation period, the cell culture medium was removed and replaced with the samples. A medium without any samples during each incubation period served as a positive control. Conversely, a medium containing a final concentration of 10% of DMSO was utilized as a negative control. After an additional 24 h of incubation, PrestoBlue (PB) reagent was introduced to the wells and changes in cell viability were detected using fluorescence spectroscopy (Synergy H1, BioTek, California, USA). After a 2-h incubation period, the fluorescence was measured (λ excitation = 570 nm; λ emission = 610 nm).
6
Phage Adsorption Kinetics in C. jejuni
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Phage adsorption assays were performed as previously described (Baldvinsson et al., 2014 (link)). Briefly, C. jejuni cultures were washed and adjusted to an OD600 of 0.4 in CBHI and inoculated with phage F341 at an MOI of 0.001. Cultures were incubated at 37°C and samples containing free phages were collected at 0, 30, 60, and 90 min and filtered through a 0.22 μm sterile syringe filter (Millipore). Filtered samples were stored at 4°C until enumeration (plaque assay). Free phages were enumerated on C. jejuni NCTC12658 and calculated as the percentage of free phages compared to time point zero for each sample (phage PFU/mL at time zero was accounted as 100%). The data are representative of two independent experiments and represent the mean percentages of free phages and standard deviations thereof.
7
Conditioned Media from AT-MSCs
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The CM were prepared as described in our previous publication [27] . Shortly after, AT-MSC (P3) were cultured in MEM Alpha (Biowest) without FBS (Biowest). After 48 h incubation in a humidified atmosphere with 5% CO 2 at 37 • C, collected media samples were filtered through a 0.2 µm sterile syringe filter (Millipore, Burlington, MA, USA). To ensure that equal concentrations (2.25 mg/mL) of CM were used for the subsequent experiments, the protein concentration of the CM was quantified via Bradford protein assay using standard Bradford reagent (Sigma). As a control (nonconditioned medium), MEM Alpha was regarded. Samples of ATMSC-CM were collected and stored at -80 • C until use.
8
Exosome Isolation for NSCLC Cell Lines and Patients
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For exosome isolation from NSCLC cell lines, ExoQuick exosome precipitation solution was added to the cell culture media, and the isolation of exosomes was conducted following the manufacturer’s instructions (SBI System Biosciences, San Francisco, CA, USA). BCA protein quantization was utilized to quantify isolated exosomes.
For exosome isolation from blood samples of NSCLC patients, all samples were subjected to centrifugation at 3,000 g for 15 min to separate plasma from cells or debris. Then an additional centrifuge at 12,000 g for 20 min was performed and the supernatant was further filtered through a sterile syringe filter (0.22 mm, Millipore, Billerica, MA, USA) into individual aliquots. Each plasma sample was mixed with an Exosome Precipitation Solution (3D Medicines biotechnology, Shanghai, China) at 4:1 v/v. The mixture was incubated for 20 min at 20°C and then centrifuged at 6,500 g for 20 min at 4°C. The liquid phase was discarded and the remaining content was further centrifuged at 1,500 g for 5 min at 4°C to remove residual liquid. Finally, the exosome pellet was obtained for immediate use.
For exosome isolation from blood samples of NSCLC patients, all samples were subjected to centrifugation at 3,000 g for 15 min to separate plasma from cells or debris. Then an additional centrifuge at 12,000 g for 20 min was performed and the supernatant was further filtered through a sterile syringe filter (0.22 mm, Millipore, Billerica, MA, USA) into individual aliquots. Each plasma sample was mixed with an Exosome Precipitation Solution (3D Medicines biotechnology, Shanghai, China) at 4:1 v/v. The mixture was incubated for 20 min at 20°C and then centrifuged at 6,500 g for 20 min at 4°C. The liquid phase was discarded and the remaining content was further centrifuged at 1,500 g for 5 min at 4°C to remove residual liquid. Finally, the exosome pellet was obtained for immediate use.
9
Preparation and Characterization of Mesenchymal Stem Cell Conditioned Media
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AT-MSCCM and UC-MSCCM at passage 3 were cultured in Dulbecco's Modified Eagle Medium (DMEM, Biowest, USA) without FBS (Biowest, USA) and antibiotics. After 24 h incubation in a humidified atmosphere with 5% CO 2 at 37°C, collected media samples were centrifuged at 400 × g for 10 min to remove cell debris, and filtered through a 0.2 µm sterile syringe filter (Millipore, USA). We have used identical procedure as published recently (Humenik et al. 2019) (link). After obtaining conditioned media from both sources, the protein concentration of the CM was quantified by Bradford protein assay, using standard Bradford reagent (Sigma), to ensure that equal concentrations (1.0 mg/ml) of CM were used. DMEM was regarded as a control (nonconditioned medium). Samples of AT-MSCCM and UC-MSCCM were collected and stored at -80°C until the time of use.
10
Blue Light Induction of E. coli Protein
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An overnight culture of E. coli containing the appropriate plasmid was used to inoculate 2 x 500mL of LB (1:200 dilution).
Once the OD600 of the cultures had reached 0.6 they were separated in two. The first flask was incubated with shaking at 18°C overnight in a darkroom (to exclude light). The second was incubated with shaking at 18°C overnight, under blue light (Supplementary Figure 1D). Each culture was harvested by centrifugation at 4000rpm and the pellets were frozen at -80°C. Pellets were lysed using lysis buffer (Imidazole 400 mM, 0.5 M NaCl, 50 mM Tris-HCl, pH 8.0) in the dark. The resulting lysate was separated from cell debris by centrifugation and filtered using a sterile syringe filter (Millipore UK). Proteins were analyzed by SDS-PAGE and western blot as detailed below.
Once the OD600 of the cultures had reached 0.6 they were separated in two. The first flask was incubated with shaking at 18°C overnight in a darkroom (to exclude light). The second was incubated with shaking at 18°C overnight, under blue light (Supplementary Figure 1D). Each culture was harvested by centrifugation at 4000rpm and the pellets were frozen at -80°C. Pellets were lysed using lysis buffer (Imidazole 400 mM, 0.5 M NaCl, 50 mM Tris-HCl, pH 8.0) in the dark. The resulting lysate was separated from cell debris by centrifugation and filtered using a sterile syringe filter (Millipore UK). Proteins were analyzed by SDS-PAGE and western blot as detailed below.
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