Extraction of the plant samples were carried out as described previously41 (link). Flavonoids were determined either as aglycones or as flavonol glycosides by preparing acid-hydrolyzed or non-hydrolyzed extracts respectively. Hydrolysis of samples was carried out as described previously42 . Briefly, for flavonol detection and quantification, plant material (1 gm) were ground into the fine powder in liquid N2 and were extracted with 80% methanol overnight at room temperature with brief agitation. The extract was filtered and filtrate was evaporated to about 1 ml. Three volume of HCl (1 M) was added into the concentrated extract and the mixture was incubated at 94°C for two h to hydrolyze any conjugate forms of flavonoids. After hydrolysis, samples were extracted with ethyl acetate, evaporated to dry and resuspended in 1 ml 80% methanol. Extracts were filtered through 0.2 μm filter (Millipore, USA) before HPLC. For non-hydrolyzed extracts, samples were extracted as described previously43 with slight modification. Briefly, plant material (1 gm) was ground into fine powder in the liquid N2 and was extracted with methanol: water (80:20) overnight at room temperature with brief agitation. The extract was filtered and filtrate was evaporated. The residue was dissolved in 1 ml methanol. Extracts were filtered through 0.2 μm filter (Millipore, USA) before analysis.
0.2 μm filter
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom, Italy
The 0.2 μm filter is a laboratory filtration device designed to remove particles and contaminants from liquids. It features a 0.2 micron pore size, which is effective at trapping microorganisms and other small particulates. The filter is constructed with durable materials suitable for use in various laboratory applications.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions)
Bovine α thrombin, by Enzyme Research (3 mentions)
Sw40 rotor, by Beckman Coulter (2 mentions)
Vivaspin 6 5000 mwco tubes, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Merck Group (2 mentions)
Streptomycin, by Merck Group (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Milli q water, by Merck Group (2 mentions)
Miracloth, by Merck Group (2 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (2 mentions)
Exosome depleted fbs, by System Biosciences (2 mentions)
Exoquick exosome precipitation solution, by System Biosciences (2 mentions)
Duoset, by R&D Systems (1 mentions)
Ethanol, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Lysozyme, by Merck Group (1 mentions)
Polydatin, by Merck Group (1 mentions)
Agilent 1200 hplc instrument, by Agilent Technologies (1 mentions)
Rutin, by Merck Group (1 mentions)
Pure acetic acid, by Merck Group (1 mentions)
100 protocols using 0.2 μm filter
1
Extraction and Quantification of Plant Flavonoids
2
Synthesis and Purification of HA-Dopamine Conjugates
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HA-Dop with a DS of dopamine relative to HA dimeric units (DS) of 5 and 10% was synthesized as previously described [22 (link)]. The molecular weight of HA in the range of 130–300 kDa was selected based on previous reports on catechol conjugation to HA [22 (link),34 (link),35 (link)]. Briefly, to obtain the HA-Dop with a DS of 10%, 250 mg of HA was solubilized in 25 mL dH2O under magnetic stirring, followed by the addition of 3 HA-COO- equivalents of EDC and 1.3 sNHS equivalents. Thirty minutes later, 1.3 equivalents of dopamine were added and left overnight to react. The reaction was maintained at pH 5.5 by adding NaOH or HCl dropwise. Dialysis over 48 h against PBS at pH 6 and dH2O was used to purify the HA-Dop conjugates from the unreacted components. After dialysis, the mixture was freeze-dried and stored at +4 °C until further use. To obtain sterile HA-Dop for in vivo implantation in rats, the same procedure was followed, and the obtained conjugate was passed through a 0.2 μm filter (Millipore Sigma, Burlington, MA, USA) before freeze-drying. To obtain HA-Dop with DS 5%, the same procedure was followed, with 2, 1.2, and 1.2 equivalents of EDC, sNHS, and dopamine, respectively.
3
Interaction of F. nucleatum with Colon Cancer Cells
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As previously described (30 (link)), HCT116 (ATCC CCL-247) cells were grown on tissue culture-treated plates and flasks in McCoy’s 5A medium supplemented with 10% fetal bovine serum (FBS), penicillin, and streptomycin. HCT116 cells were seeded to confluence in 24-well plates (2 × 105 cells per well at 100% confluence), and F. nucleatum subspecies were added at a multiplicity of infection (MOI) of 50:1 followed by incubation at 37°C and 5% CO2 for 4 h. Medium from individual wells was sterile filtered using a 0.2-μm filter (MilliporeSigma) and diluted to concentrations within the range of the R&D Systems DuoSet enzyme-linked immunosorbent assay (ELISA) to analyze human IL-8 and CXCL1 concentrations.
4
Microfluidic Spheroid Formation Protocol
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Before introducing the cells to the devices, by using 99.9% ethanol (Sigma-Aldrich, St. Louis, MO, USA), microfluidic channels were sterilized and the air bubbles were removed from the channels. To reduce cell adhesion to the PDMS surfaces and promote the spheroid formation, we performed surface coating with BSA. BSA (Sigma-Aldrich) solutions in two different concentrations of 3% w/v and 10% w/v in sterile phosphate buffered saline (PBS, Sigma Aldrich) were prepared [27 (link)] and sterilized by filtration through a 0.2 μm filter (Millipore Sigma). The microfluidic channels were flushed out 3–4 times with the BSA solutions to remove the ethanol residual.
Then, by incubation of freshly prepared BSA solutions with biochips, their surfaces were modified to assess spheroid formation on-chip. One group of biochips was surface treated with 3% BSA, and another group of biochips was surface treated with 10% BSA to assess the impact of surface treatment on spheroid production. Only 10% BSA was used for co-culture model assays. All the devices were incubated overnight in the incubator (37 °C, 5% CO2, 95% ambient air) right after the cell seeding process.
Then, by incubation of freshly prepared BSA solutions with biochips, their surfaces were modified to assess spheroid formation on-chip. One group of biochips was surface treated with 3% BSA, and another group of biochips was surface treated with 10% BSA to assess the impact of surface treatment on spheroid production. Only 10% BSA was used for co-culture model assays. All the devices were incubated overnight in the incubator (37 °C, 5% CO2, 95% ambient air) right after the cell seeding process.
5
Screening Antimicrobial Activity of Probiotic Strains
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Bifidobacterium, Enterococcus, and Lactococcus strains were (Table 1 ) streaked on freshly prepared MRS or brain-heart infusion (BHI) agar plates. After overnight incubation under anaerobic conditions at 37°C, a single isolated colony was inoculated into fresh MRS/BHI broth and grown for 12–16 h without shaking at 37°C. The resulting culture was subcultured (4–5%, vol/vol) overnight in fresh medium, followed by growth under the aforementioned conditions. After 4–5 h of growth, the culture was subjected to centrifugation for 10 min at 3,000 g to harvest the cells. The resulting supernatant was collected, and passed through a 0.2-μm filter (Millipore, Bangalore, India) and based on the necessity it was concentrated by using Millipore concentrators with a 5-kDa molecular weight cutoff. The resulting concentrated supernatant was subjected to a well diffusion, disk diffusion, or unique well diffusion assay (UWDA; our method).
6
Recombinant CD155 D2/sub-D2 Expression
7
Isolation and Characterization of Extracellular Vesicles
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Cells
at passage 1–20
were cultured until 80% confluency in their complete medium and then
in DMEM containing 5% depleted FBS for 48 h. Next, the conditioned
media from ∼107 cells were collected, filtered through
a 0.2 μm filter (Millipore), and centrifuged at 300g for 10 min. The supernatant was concentrated to 0.5 mL via a centrifugal
filter (Centricon Plus-70 centrifugal filter 10 kDa cutoff) and added
to a qEV 70 nm column (iZon). As the supernatant passed through the
column, 4 mL of PBS was added in 100–200 μL aliquots.
First, 3 mL was discarded, and a subsequent 1.5 mL was collected as
an EV fraction. As a final step, EVs were concentrated with an Amicon
Ultra-2 centrifugal filter unit with an Ultracel-100 membrane. The
size and concentration of prepared EVs were measured via nanoparticle-tracking
analysis (LM10, Melvern).
at passage 1–20
were cultured until 80% confluency in their complete medium and then
in DMEM containing 5% depleted FBS for 48 h. Next, the conditioned
media from ∼107 cells were collected, filtered through
a 0.2 μm filter (Millipore), and centrifuged at 300g for 10 min. The supernatant was concentrated to 0.5 mL via a centrifugal
filter (Centricon Plus-70 centrifugal filter 10 kDa cutoff) and added
to a qEV 70 nm column (iZon). As the supernatant passed through the
column, 4 mL of PBS was added in 100–200 μL aliquots.
First, 3 mL was discarded, and a subsequent 1.5 mL was collected as
an EV fraction. As a final step, EVs were concentrated with an Amicon
Ultra-2 centrifugal filter unit with an Ultracel-100 membrane. The
size and concentration of prepared EVs were measured via nanoparticle-tracking
analysis (LM10, Melvern).
8
Standardized Extraction and Analysis of Polygonum cuspidatum
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The stem component of P. cuspidatum was provided by Samil. Co. Ltd (Seoul, Korea). Briefly, P. cuspidatum (700 g) was extracted in distilled water by incubating at 100°C for 4 h, followed by freeze-drying (yield: 6.57%). The PSE was standardized using the reference compounds, polydatin and rutin (Sigma, MO, USA) by high-performance liquid chromatography (HPLC) according to previously described protocols12 (link). Briefly, PSE (10 mg) was dissolved in 50% methanol (10 mL). The solution was filtered through a 0.2 μm filter (Millipore, MA, USA) prior to injection. HPLC analysis was performed with an Agilent 1200 HPLC instrument (Agilent Technologies, CA, USA) equipped with a binary pump, vacuum degasser, auto-sampler, column compartment, and diode array detector.
9
Cigarette Smoke Extract Generation
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CSE was generated by lighting four Marlboro Reds, Class A cigarettes, through 30 mL of Roswell Park Memorial Institute 1640 (RPMI) (Lonza, Switzerland). The generation of CSE is based on a validated pump system.13 Each cigarette was smoked for 10 puffs, each puff generating 35 mL of smoke, every 30 s, which burned approximately 75% of the cigarette. The volume of smoke generated was 350 mL. Each milliliter of CSE contains 0.133 (4/30) cigarette's worth of smoke‐derived constituents. The resultant CSE was sterile‐filtered through a 0.2‐μm filter (Millipore), with pH adjusted to 7.4, and stored at −20°C. The CSE was diluted in RPMI to make 5%, 10%, and 20% solutions that were used for ex vivo human explant tissue experiments. Based on Bernhard's validated volumetric calculations,13 a 5% solution of CSE equates to 40 cigarettes per day.
10
Modeling Helminth Infection in Mice
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BALB/c and MIF-deficient mice on the BALB/c background were bred in-house and housed in individually-ventilated cages (IVCs) according to UK Home Office guidelines. The Mif−/− construct contains a neomycin cassette disrupting exons 2 and 3 of the Mif gene75 (link). ACKR3-GFP mice (Ackr3tm1Litt)82 (link) were kindly provided by Prof. Gerard Graham, University of Glasgow, UK.
Infections employed N. brasiliensis maintained as previously described83 . NES was collected as spent culture medium collected over 7 days from adult N brasiliensis as described84 (link), centrifuged at 400 g for 10 min to remove eggs, and diafiltrated into PBS over a 3000 MW cut-off membrane in an Amicon, sterilized by passage through a 0.2 μm filter (Millipore) and frozen at −80 °C.
Adult worm counts were conducted after small intestines were removed and sliced longitudinally. Egg counts were performed on 3–4 fecal pellets which were weighed and resuspended in 2 ml dH2O; 2 ml of saturated salt solution (400 g NaCl in 1 L dH2O) was then added and eggs enumerated using a McMaster egg counting chamber. Egg counts are represented as eggs/g fecal material.
Infections employed N. brasiliensis maintained as previously described83 . NES was collected as spent culture medium collected over 7 days from adult N brasiliensis as described84 (link), centrifuged at 400 g for 10 min to remove eggs, and diafiltrated into PBS over a 3000 MW cut-off membrane in an Amicon, sterilized by passage through a 0.2 μm filter (Millipore) and frozen at −80 °C.
Adult worm counts were conducted after small intestines were removed and sliced longitudinally. Egg counts were performed on 3–4 fecal pellets which were weighed and resuspended in 2 ml dH2O; 2 ml of saturated salt solution (400 g NaCl in 1 L dH2O) was then added and eggs enumerated using a McMaster egg counting chamber. Egg counts are represented as eggs/g fecal material.
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