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There are currently a total of 27 assays in ViNAS.
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| NanoAID | Name | Measurement | Description | Control | Note |
|---|---|---|---|---|---|
| 1 | AChE Binding 1 | Quenching of AChE intrinsic fluorescence | Steady state fluorescence spectra were measured using a Hitachi F-4500 spectrofluorometer. AChE solution concentration was 0.36 μM in 0.1 mM PBS buffer solution. Intrinsic fluorescence of AChE was measured by addition of nanoparticles stock solutions of which the final concentration was 90.9 μg/ml. AChE solutions were excited at 280 nm and emission wavelength was set from 300 to 400 nm. Scanning speed was 1200 nm/min. Excitation and emission slit was set to 10.0 and 5.0 nm, respectively. PMT voltage was set to 700 volt. Fluorescence intensities at 340 nm were used for calculating the fluorescence queching effect. All measurements were performed at room temperature (23℃). We transformed the fluorescence values with and without the nanoparticles were transformed according to the Stern-Volmer equation: F₀/F = 1 + Ksv[Q], where F₀ and F are the fluorescence intensities in the absence and presence of the quencher, and Ksv is the dynamic quenching constant. | Cell culture medium | - |
| 2 | AChE Binding 2 | Inhibition of AChE activity (%) | The asay buffer was 100 mM PBS, PH=8.0. A stock solution of AChE (100 U/ml) in assay buffer was kept at 0℃. A 1:30 dilution was prepared immediately before starting the measurement. ATCh (10 mM) and DTNB (7 mM) were dissolved inassay buffer and kept at 0℃. Stock solution concentration of nanoparticles dissolved in PBS was 1mg/ml. Neostigmine bromide, a known competitive inhibitor of AChE, was used as positive control and the concentration of stock solution was 0.1 mM. Into a cuvette containing 880 μl of assay buffer, 50 μl of the DTNB solution, 10 μl of an inhibitor solution, and 10 μl of an AChE solution (3.33 U/ml) were added and thoroughly mixed. After incubation for 15 min at 25 ℃, the reaction was inhibited by adding 50 μl of ATCh solution. The absorbance were monitored at 412 nm over 5 min. The inhibition rates were calculated using the equation i(%) = (1-v/v₀)×100%, where v₀ and v are the rates in the absebce and presence of inhibitor. | Negative control: cell culture medium; Positive control: Neostigmine bromide | - |
| 3 | Autophage | Autophage inducing ability (number of the green fluorescent puncta per cell) | Tested in triplicate. The LC3-GFP U87 repoter cells were seeded in confocal dishes and fixed with 4% paraformaldehyde. Laser scanning confocal microscopy was used to acquire fluorescent images of cells. To quantify cell autophagy induction, the number of bright punctuates (autophagosomes) was counted in at least 30 cells. | Negative control: cell culture medium; Positive control: Rapamycin | - |
| 4 | Cell Association | Cellular association in A549 cell (Mg, log2 transformed) | Tested in triplicate. For cell association studies, harvested A549 cells were plated onto 24 well plates at ~200000 cells/well and incubated overnight at 37℃ to reach ~80% confluence. Nanoparticles were incubated with cells for 4 h at 37℃. Following inclubation, cells in each well were washed four times with sterile PBS supplemented with 0.133 g/l calcium chloride dihydrate and 0.1% bovine serum albumin to remove particles that were free in solution and/or not strongly assoicated with the cell surface.Total cell association (y) was calculated using the following pseudopartition coefficient: y = mcell/(mwell × mcells). Where, mcell is the total atomic golf (or silver) content associated with cells, mwell is the total atomic gold (or silver) content in well (associated with cells and free in solution), and mcells is the total mass of magnesium per sample. | NaN | - |
| 5 | Cell Uptake in A549 Cells | Cellular uptake in A549 (1×10⁻¹¹ g Au cell⁻¹) | Tested in triplicate. Nanoparticles (50 μg/ml) were incubated with A549 cells for 24 h. After washing cells three times with phosphate buffered saline, we detached the cells from flask by trypsin-EDTA solution. The cells were counted and then lysed overnight in aqua regia. ICP-MS was used to quantify the concentration of nanoparticles. | Cell culture medium | - |
| 6 | Cell Uptake in A549 Cells 2 | Cellular uptake in A549 (1×10⁶ nm² cell⁻¹) | Tested in triplicate. A549 cells were seeded in 24-well plates at a density of 100 000 cells/well. After 24 h, the cells were washed once with PBS, and the solutions of nanoparticles in cell culture medium (2.5×10¹⁴ nm²/ml) were added. After incubation for 12 h, the samples were washed seven times with PBS to remove extra nanoparticles. Then,the cells were detached by trypsin−EDTA solution (0.25% trypsin, 1 mM EDTA) and counted. The detached cells were lysed for ICP-MS. | Cell culture medium | - |
| 7 | Cell Uptake In HEK293 Cells | Cellular uptake in HEK293 (1×10⁻¹¹ g Au cell⁻¹) | Tested in triplicate. Nanoparticles (50 μg/ml) were incubated with HEK293 cells for 24 h. After washing cells three times with phosphate buffered saline, we detached the cells from flask by trypsin-EDTA solution. The cells were counted and then lysed overnight in aqua regia. ICP-MS was used to quantify the concentration of nanoparticles. | Cell culture medium | - |
| 8 | Cell Viability | Cell viability (200 μg/ml) | Tested in triplicate. THP-1 (human monocyte) cell lines were cultivated in RPMI 1640 with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 100 μg/ml penicillin and 100 U/ml streptomtcin and grown in a humidified incubator at 37℃. Cell differentiation into macrophages was triggered by adding Phorbol 12-myristate 13-acetate at a concentration of 50 ng/ml and incubating for 48 h.Differentiated cells were characterized by allowing them to adhere to the plastic well surface in 96 well plates. The nonadherent monocytes were removed, and the adherent macrophages were washed twice in RPMI 1640. Cells were treated with f-MWNT suspensions (50 and 200 μg/ml in complete culture medium. LPS was added to the cultures at a concentration of 100 ng/ml. After 24 h of incubation, a cell proliferation (WAT-1) assay was used to determine the cell viability. | Negative control: Cell culture medium; Positive control: Lipopolysaccharide (LPS) | - |
| 9 | logP | logP | Tested in triplicate. The experimental logP values of all the nanoparticles were determined using "shaking flask" method. Briefly, nanoparticles were mixed with octanol-saturated water and water-saturated octanol. The mixture was shaken for 24 h. Then, the mixture was kept still for 3 h to seperate the organic and water phases. The nanoparticles in both phases were quantitatively determined by ICP-MS. logP values were then calculated using the following equation: logP = log[Cnp(octanol)/Cnp(water)]. Where, Cnp(octanol) is the concentration of nanoparticles in octanol and Cnp(water) is the concentration of nanoparticles in water. | NaN | - |
| 10 | Metabolic Activity of CYP34A | Metabolic activity of CYP3A4 in the liver (%) | The CYP3A4 activity in the HLM-only group was defined as 100%, and that in the ketoconazole group was defined as 0%. The activity of CYP3A4 in functional CNT treated groups was calculated according to the following equation: CNT's effect on CYP3A4 activity = (peak area of NFP in ketoconazole group - peak area of NFP in CNT group)/ (peak area of NFP in ketoconazole group - peak area of NFP in HLM-only group). | Negative control: Human liver microsomes (HLM); Positive control: ketoconazole | - |