For tumor cell transendothelial migration assay, HUVEC cells (5 × 104 cells per well) were added to the transwell cell culture chambers with polycarbonate membranes of 8.0-mm diameter pores (Millipore). Transfected HCT116 and SW480 cells were plated on their upper side for 24 hours and fixed in 4% paraformaldehyde. HUVEC were stained with anti-CD31 (Bioss) antibody. Transwell filters were examined by using a laser confocal microscope (Olympus FV1000). For migration and invasion assay, transfected HCT116 cells (1 × 105 cells per well) were seeded on the transwell cell culture chambers with polycarbonate membranes of 8.0-mm diameter pores (Millipore) for 24 hours, fixed in 4% paraformaldehyde for 10 min, and stained using crystal violet solution for 30 min. Stained cells were washed with water gently. Images were captured and quantified by ImageJ.
Polycarbonate membrane
Manufactured by Merck Group
Sourced in United States, Germany, Ireland, United Kingdom
Polycarbonate membranes are a type of filtration media used in various laboratory applications. They are made from a durable, transparent polymer material that provides efficient size-based separation of particles, molecules, and cells. These membranes are characterized by their precise and uniform pore sizes, which enable precise filtration and isolation of target analytes. Polycarbonate membranes are commonly used in techniques such as flow cytometry, microfiltration, and sample preparation.
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Lab products found in correlation
Matrigel, by BD (5 mentions)
Streptomycin, by Thermo Fisher Scientific (4 mentions)
100cx 2 tem, by JEOL (4 mentions)
Penicillin, by Thermo Fisher Scientific (3 mentions)
Sodium chloride (nacl), by Merck Group (3 mentions)
Dspe peg2000, by NOF corporation (3 mentions)
Cholesterol chol, by Merck Group (2 mentions)
Gelatin, by Merck Group (2 mentions)
Dubelcco s modified eagle s medium dmem, by Merck Group (2 mentions)
Amphotericin b psa, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum fsb, by Thermo Fisher Scientific (2 mentions)
Hepes, by Merck Group (2 mentions)
Cholesteryl hemisuccinate, by Merck Group (2 mentions)
Mda mb 231, by American Type Culture Collection (2 mentions)
Zetasizer nano series, by Malvern Panalytical (2 mentions)
Hepes salt, by Merck Group (1 mentions)
Trypsin edta 0.25, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Chemotactic chamber, by Neuro Probe (1 mentions)
71 protocols using polycarbonate membrane
1
Tumor Cell Transendothelial Migration Assay
2
Cell Migration Assay Protocol
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For cell pretreatment, refer to the wound-healing assay section. The assay was carried out within the 24-well Boyden chamber coated with the polycarbonate membrane (with the pore size of 8-µm, Millipore, Billerica, MA, USA) as well as Matrigel for the formation of the matrix barrier. RPMI 1640 medium would be put into bottom chamber with 10% FBS to be the chemoattractant. Afterwards, the cells were cultured for 24 h in the incubator. In addition, those that were still staying on the internal channel membrane would be eliminated by the cotton swab, whereas those adhering to the lower membranes would be subjected to methanol fixation and crystal violet staining. Afterwards, the migrating cell number in each microscopic field of view (FOV) was recorded and examined.
3
Cyanobacterial Bloom Monitoring in Lake Erie
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Two drinking water treatment plants in Ohio, United States, were selected based on annual cyanobacterial bloom intensity. The first was the Collins Park Water Treatment Plant in Toledo, which is a historically high-bloom area in western Lake Erie (Ohio Department Health et al., 2020 ) and the second was the City of Painesville Water Treatment Plant, a low- or no-bloom area in central Lake Erie (Supplementary Figure S1 ). Water samples were collected once weekly during the summer and fall (May–November) of 2013 and 2014 from the Toledo and Painesville sites, totaling 58 and 60 samples, respectively. Samples were collected from the source water (sampled at the drinking water intakes within Lake Erie) and from the finished drinking water at each plant. Collections were done in sterile bottles and shipped in a cooler on ice. For toxin measurements, amber glass vials were used. The samples reached The Ohio State University (Columbus, Ohio, United States) for laboratory analysis within 15 h of sample collection. In the lab, the collected source water (200 mL) was filtered using the polycarbonate membrane (0.45 μm pore size, Millipore, Burlington, MA, United States) for molecular analyses, and filters were kept at −20°C and further analyses were performed as soon as possible.
4
Functionalization of Multi-Walled Carbon Nanotubes
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In order to functionalize multi-walled carbon nanotubes, an appropriate amount of MWCNT was dispersed in a solution of concentred H2SO4 and HNO3, with a volume ratio of 1:3. The suspension was ultrasonicated for a few minutes, then left under magnetic stirring over-night. Afterwards, the functionalized multi walled carbon nanotubes (f-MWCNT) were filtered using a Millipore polycarbonate membrane with a diameter of 0.22 µm, and washed with bi-distillated water until a neutral pH of the filtrate was obtained. Eventually, the f-MWCNT were dried at the oven at 50°C for 5 h.
5
Nanoparticle Formulation for Tumor Targeting
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DOX was supplied by Lancrix Chemicals (Shanghai, China), with a purity higher than 99.9%. Hydrogenated soy phosphatidylcholine (HSPC), egg phosphatidylglycerol (EPG), and Distearoyl-phosphoethanolamine-polyethilenglycol2000 (DSPE-PEG2000) were acquired from Lipoid GmbH (Ludwigshafen, Germany). Cholesterol (CHOL) and HEPES salt were supplied by Sigma Chemical Company (St. Louis, MO, USA). The polycarbonate membrane was purchased from Millipore (Billerica, MA, USA). All other compounds and solvents used in this work were of analytical grade. RPMI 1640 Medium, fetal bovine serum (FBS), penicillin, streptomycin, and trypsin EDTA 0.25% were purchased from Gibco-Invitrogen (Waltham, MA, USA). The subcutaneous tumor model was established in 7–8-week female BALB/c mice purchased from CEBIO-UFMG (Belo Horizonte, Brazil).
6
Preparation of Multilamellar and Unilamellar Lipid Vesicles
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Multilamellar vesicles (MLVs), containing a specific molar ratio of selected lipids, were prepared to a final concentration of 5 mg/mL of total lipids. Each lipid film was prepared by evaporation of organic solvents under reduced pressure and hydrated with Tris (20 mM Tris–HCl, 140 mM NaCl, 1 mM EDTA, pH 8.0) or Hepes (10 mM HEPES, 150 mM NaCl, pH 7.4) buffer and vortexed extensively to gain MLVs43 (link). Large unilamellar vesicles (LUVs) composed of CPE/POPC/cholesterol (molar ratio, 1:1:1) were prepared from MLVs, by 8-times snap freezing the vesicles in liquid nitrogen, each time followed by thawing at 60°C, and extruded through the polycarbonate membrane with 100 nm pores at ~ 50°C (Millipore, Germany). MLVs and LUVs were stored at 4°C and used within five days.
7
Caco-2 Cell Monolayer Permeability Assay
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For transport experiments, Caco-2 cells were seeded onto polycarbonate membrane (0.6 cm2 growth area, 0.4 µm pore size, Millipore, MA) at a density about 1.0 × 105 cells/cm2 and allowed to grow for 21–25 d [17] . Permeability studies were conducted with the monolayers that developed the transepithelial electrical resistance (TEER) values above 250 Ω cm2. Samples (100 µl) at 15, 30, 45, 60, 90, 120 min were taken from the basolateral (BL) solution, and the volume was replaced with prewarmed Hanks’ balanced salt solution (HBSS). Because the amino acid prodrugs were partially metabolized to peramivir and amino acids during the transport across the Caco-2 monolayers, the prodrugs transported was calculated as the total sum of the unchanged prodrugs and peramivir.
8
Microbial Community Analysis by FISH
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Sample fixation and FISH procedures followed previously described methods39 . Samples were fixed for 12 hours with 2% formaldehyde, washed twice with PBS, and finally re-suspended with a 1:1 mix of PBS/ethanol. Fixed samples were filtered through a polycarbonate membrane with a 0.22 μm pore size (Millipore, Eschborn, Germany). Then, the membrane was subjected to hybridization and washing procedures as described previously39 . Probes specific to ANME-2 (ANME-2-538-Alexa594 and ANME-2-538-AMCA)40 (link), Desulfobacteriaceae (DSS658-Alexa488)41 , and Betaproteobacteria (BET-42a-Alexa594)42 were purchased from Life Technologies (Shanghai, China). For FISH with multiple probes, a formamide concentration of 50% was used for both DSS658 and ANME-2-538, and of 35% for BET-42a.
9
Cisplatin Release from Liposome Formulations
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The concentration of cisplatin, iron, phospholipids, and cholesterol and values of ζ-potential were measured 15 days and 1 month after preparation of the liposome suspensions. They were stored at 4 °C and protected from light.
Cisplatin release from the Cis-MLs was assessed by using Franz diffusion cells assembled with a polycarbonate membrane (pores 0.05 µm) (Millipore Co., County Cork, Ireland) at room temperature in saline solution and at 37 °C in human serum. An aliquot of Cis-MLs was placed into the donor cell compartment and tamped down to the polycarbonate membrane. At predetermined time intervals from 1 to 48 h, the whole receptor phase solution (5–6 mL) was withdrawn and the cisplatin concentration was evaluated by HPLC [49 (link)]. At each time point, the percentage of free cisplatin was calculated with the following equation:
where Wt is the amount in the receptor phase and W0 is the initial amount of cisplatin placed in the donor phase.
Cisplatin release from the Cis-MLs was assessed by using Franz diffusion cells assembled with a polycarbonate membrane (pores 0.05 µm) (Millipore Co., County Cork, Ireland) at room temperature in saline solution and at 37 °C in human serum. An aliquot of Cis-MLs was placed into the donor cell compartment and tamped down to the polycarbonate membrane. At predetermined time intervals from 1 to 48 h, the whole receptor phase solution (5–6 mL) was withdrawn and the cisplatin concentration was evaluated by HPLC [49 (link)]. At each time point, the percentage of free cisplatin was calculated with the following equation:
where Wt is the amount in the receptor phase and W0 is the initial amount of cisplatin placed in the donor phase.
10
Chemotaxis Assay for Monocyte Migration
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Chemotaxis was analysed as previously described [21 (link)]. Briefly, freshly isolated human monocytes or cryopreserved monocytes were resuspended in medium at a concentration of 0.5 × 106 cells/ml. Subsequently, monocytes were placed to the upper wells of a chemotactic chamber (Neuroprobe) and migrated towards chemotactic growth factors which were added to the lower wells of the chamber. Upper and lower wells were separated by a polycarbonate membrane (pore size 5 μm, Millipore). The cells were allowed to migrate for 90 min in a humidified incubator (5 % CO2) at 37 °C. Adherent cells on polycarbonate membrane were fixed for 10 min using absolute ethanol and stained with Giemsa dye. The non-migrated cells from the upper side of the membrane were scraped off gently with a cotton bud. Migrated cells were quantified by counting cells in five high-power fields (20× primary magnification) of four different wells per condition.
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