At the end of the predetermined proximity time of cells with the sealer extract, 10 µl of MTT solution was added to each of the wells of the plate and placed in the incubator for 2–4 hr. The culture medium on the cells was drained, and 50 µl of dimethyl sulfoxide (DMSO) (Merk-Germany) was added to each well to dissolve the reduced formazan dye. The intensity of the resulting color was then evaluated by the light absorption of each well using the Elisa reader (Awareness Technology Inc.) at a wavelength of 545 nm (with a reference of 630 nm) [12 (link), 13 (link)].
ELISA reader
Manufactured by Awareness Technology
Sourced in United States
The ELISA reader is a laboratory instrument used to measure the optical density of samples in enzyme-linked immunosorbent assay (ELISA) experiments. It functions by detecting and quantifying the level of a specific substance, such as a protein or antibody, in a liquid sample.
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11 protocols using ELISA reader
1
MTT Assay for Cell Viability
2
Inflammatory Marker Assessment via ELISA
Inflammatory markers were measured using enzyme-like immune-sorbent assay (ELISA reader, awareness technology, USA). All variables were measured with kits obtained from Diaclone, Besançon, France. Serum concentrations were performed for IL-1β with the sensitivity of the kit 6.5 pg/mL. The sensitivity of IL-6 was 2 pg/mL and average inter- and intra-assay coefficients of variability (CVs) were 7.7 and 3.6%, respectively. TNF-α had a sensitivity 8 pg/mL and average inter- and intra-assay CVs were 10.9 and 3.2%, respectively. All analyses were performed in duplicates.
3
Cytotoxicity Assay of DOX-FGO Formulations
For the measurement of cytotoxicity, an MTT assay was used.33 (link)
The MG-63 cells were cultured in RPMI medium (containing streptomycin and penicillin) with 10% FBS and incubated at 37°C (with 95% humidity and 5% CO2). To assay cytotoxicity, 10 000 cells were seeded per well in the 96 well plates, and 200 µL of culture medium was added with 10% FBS to each well.34 (link)
The plates were incubated at 5% CO2 and in a humidified atmosphere for 24 hours at 37°C so that the cells have enough time to stick to the bottom of the wells. After preparing serial dilutions, the cells were treated with indicated concentrations of FGO, free DOX, and DOX-FGO (10, 5, 2.5, 1.25, 0.625, 0.312 µg/ mL). The concentrations of DOX-FGO were based on the content of DOX. A control group without treatment for each of these groups was considered. Each of the wells was washed two times with PBS after 48 hours of incubation. Then, a 180 µL growth medium with 10% FBS and 20 µL MTT solution was added to each well and incubated for 4 hours. After this period, the contents were removed and 150 µL DMSO was added to each well to solve the crystals of formazan. ELISA reader (Awareness Technology, Palm City, FL, USA) at 570 nm was used to determine the absorbance of the formazan. All tests were performed in triplicate. The following formula was used to calculate the cell viability:
4
Cytotoxicity of DXL-loaded Nano-niosomes
The cytotoxicity of DXL-loaded nano-niosomes was investigated by the MTT assay and compared to free drug against MDA-MB-231, Calu-6, and AsPC-1 cell lines.
The seeded cells in 96-well plates (2×104 per well, in Dulbecco’s Modified Eagle Medium (DMEM) contained 10% FBS, and 1% streptomycin and penicillin) were treated with different concentrations
of DXL-loaded nano-niosomes (1, 2, 5, 10, 20, 50 and 100 nmol L-1). Untreated cells were considered as a control.
The plates were incubated for 48 h at 37 °C in a humid atmosphere with 5% CO2. Then, 10 μL of a MTT solution (5 mg mL-1 in PBS) was added to the wells, and the plates were placed for 4 h in dark.
After that, the media were separated and 100 µL of DMSO was added to the wells for dissolving the formazan crystals. The optical density was read at 570 nm using an Awareness Technology (USA) ELISA reader. All experiments were repeated three times at sterile conditions.
The seeded cells in 96-well plates (2×104 per well, in Dulbecco’s Modified Eagle Medium (DMEM) contained 10% FBS, and 1% streptomycin and penicillin) were treated with different concentrations
of DXL-loaded nano-niosomes (1, 2, 5, 10, 20, 50 and 100 nmol L-1). Untreated cells were considered as a control.
The plates were incubated for 48 h at 37 °C in a humid atmosphere with 5% CO2. Then, 10 μL of a MTT solution (5 mg mL-1 in PBS) was added to the wells, and the plates were placed for 4 h in dark.
After that, the media were separated and 100 µL of DMSO was added to the wells for dissolving the formazan crystals. The optical density was read at 570 nm using an Awareness Technology (USA) ELISA reader. All experiments were repeated three times at sterile conditions.
5
Cytotoxicity Evaluation of Epirubicin Nanoparticles
MTT assay was used to examine the cytotoxicity of prepared nanoparticles containing epirubicin in vitro. HT-29 as an EpCAM-positive colorectal cancer and CHO as an EpCAM-negative cell line were seeded (6 × 103 cells/well) separately in 96-well plates. After 24 h incubation in a humidified 5% CO2 incubator at 37 °C, cells were treated in three groups including Free EPI, QD@MSN-EPI-Au-PEG, and QD@MSN-EPI-Au-PEG-Apt at equivalent concentrations of EPI ranging from 0.39 to 25 µg/ml for 4 h. Then, the treatment culture media were exchanged with fresh RPMI 1640 containing 10% FBS and further incubated at 37 °C for 24, 48, and 72 h. Afterward, 20 μl of MTT (5 mg/ml in PBS) was added to each well and incubated for another 4 h. After MTT reduction, the medium was aspirated and 150 µl DMSO was added to dissolve formazan crystals. In the end, the absorbance of the purple product was determined by an ELISA reader (Awareness Technology, Inc.) at 540 nm.
6
Plasma SOD Activity Quantification
SOD activity measurement in plasma was made by measuring at a wavelength of 505 nm the intensity of the violet-colored formazan dye created by reacting with iodonitrotetrazolium. SOD activity in plasma was determined using a commercially available kit as recommended by the manufacturers (Cayman Chemical, Ann Arbor, Michigan, USA). Absorbance was measure by an ELISA reader (Awareness Technology, Inc., Palm City, USA). Results are given in U/mL.
7
Quantifying Apoptosis in CLL-CII Cells
Cell death was determined with an ELISA apoptosis kit (Roche Diagnostics GmbH) that
determines monoand oligonucleosomes released into the cytoplasm of apoptotic cells (25 (link)).
The CLL-CII cells were cultivated at a density of 1×105 cells/well in 6-well
culture plates and exposed to miRNA-16-1 and ABT-199, as described
previously. After 24-48 hours of incubation, the cells were lysed and ELISA assay was
performed according to the manufacturer’s instructions. Briefly, 20 µl of the supernatants
and 80 µl of immunoreagent containing DNA-peroxidase and histone-biotin antibodies were
added to each well of a streptavidin-coated plate and the plate was incubated for 2 hours
at room temperature. After washing with incubation buffer, 100 µl of ABTS solution was
added. Finally, the reactions were stopped with ABTS stop solution and absorbance was
quantified immediately by an ELISA reader (Awareness Technology, Palm City, FL, USA) at
405 nm. Data were calculated as the fold increase in the absorbance of test groups
relative to the control group.
determines monoand oligonucleosomes released into the cytoplasm of apoptotic cells (25 (link)).
The CLL-CII cells were cultivated at a density of 1×105 cells/well in 6-well
culture plates and exposed to miRNA-16-1 and ABT-199, as described
previously. After 24-48 hours of incubation, the cells were lysed and ELISA assay was
performed according to the manufacturer’s instructions. Briefly, 20 µl of the supernatants
and 80 µl of immunoreagent containing DNA-peroxidase and histone-biotin antibodies were
added to each well of a streptavidin-coated plate and the plate was incubated for 2 hours
at room temperature. After washing with incubation buffer, 100 µl of ABTS solution was
added. Finally, the reactions were stopped with ABTS stop solution and absorbance was
quantified immediately by an ELISA reader (Awareness Technology, Palm City, FL, USA) at
405 nm. Data were calculated as the fold increase in the absorbance of test groups
relative to the control group.
8
Cytotoxicity Evaluation of Additives
The cytotoxic effects of the additives were elaborated by methyl-thiazol-tetrazolium (MTT) assay Kit (Sigma). Concisely, TE-8 cells were seeded at a density of 15×103 cells/well in 96-well culture, and then treated with the agents and incubated in the humidified CO2 incubator. Subsequently, 100 µl of MTT reagent (0.5 mg/ml in PBS) was transferred to each well and the plates were incubated for 4 h. The water-insoluble formazan crystals were constructed during the incubation period that were solubilized by adding 100 µl of the solubilization buffer [Dimethyl sulfoxide (DMSO) + Sorensen buffer] to each well. After 30 min of incubation in the conditions mentioned early, the optical density (OD) of each well was measured at 570 nm using an ELISA reader (Awareness Technology, Palm City, FL, USA). All experiments were done in triplicate order.
9
Cytokine Concentration Quantification by ELISA
ELISA kits (MABTECH, Sweden) were used based on the manufacturer’s instructions to specify the concentrations of Supernatant Cytokines (IL-18, IL-1β). The absorbance was read by an ELISA reader (Awareness Technology, FL, USA). IL-1β, IL-18 levels were expressed as pg/mL. The lowest limits of detection were, 20 pg/mL for IL-1β, 9.8 pg/mL for IL-18; respectively.
10
Microtiter Dish Biofilm Formation Assay
The phenotypic biofilm production was performed by microtiter dish biofilm formation assay. Isolates were grown in TSB for 24 hours at 37°C, then cultures was diluted 1:100 and 200 μL of the prepared inoculum was added to each well of a microplate and incubated overnight at 37°C. According to the protocol (11 (link)), eight replicates were tested for each isolate. After incubation, planktonic cells were removed by turning the plate over and shaking out the liquid, then plates were submerged three times in water and shaked out. 200 μL of 0.1% crystal violet solution was added to each well and plates were incubated for 15 minutes at room temperature, then they were submerged in a water tub for 3–4 times, shacked out, knocked on paper towels and dried. 250 μL of acetic acid solution (30% in water) was added to each well to solubilize the dye and plates were incubated at room temperature for 10 minutes. Contents were transferred to a new, flat-bottomed microtiter dish and absorbance was measured by ELISA reader (Awareness technology Inc) at 550 nm.
Biofilm producer isolates was defined as described elsewhere (12 (link)). In brief, the optical density cut-off (ODc) value is considered as: average OD of negative control + 3 × SD (standard deviation) of negative control, and the biofilm producers were classified as shown inTable 1 .
Biofilm producer isolates was defined as described elsewhere (12 (link)). In brief, the optical density cut-off (ODc) value is considered as: average OD of negative control + 3 × SD (standard deviation) of negative control, and the biofilm producers were classified as shown in
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