Cytotoxicity resulting from E. coli vectors was determined using a modified MTT assay.12 Briefly, after macrophage seeding and the 24 h incubation, media was removed and cells were combined with bacterial vectors prepared as described above except the delivery volume was 50 μL if gentamicin treatment was included or 100 μL if gentamicin treatment was excluded. Following another 24 h incubation, cells were assayed with MTT solution (5 mg/mL), added at 10% v/v, for 3 h at 37 °C/5% CO2. Medium plus MTT solution was then aspirated and replaced by DMSO to dissolve the formazan reaction products. Following agitated incubation for 1 h, the optical density of the formazan solution was analyzed using a Synergy 4 multi-mode microplate reader at 570 nm, with 630 nm serving as the reference wavelength. Results are presented as a percentage of untreated cells (100% viability).
Synergy 4
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The Synergy 4 is a multi-mode microplate reader designed for a wide range of applications. It features advanced detection technologies, including absorbance, fluorescence, and luminescence, allowing for accurate and reliable measurements of various biological and chemical samples.
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Lab products found in correlation
16 protocols using synergy 4
1
Cytotoxicity Assessment of E. coli Vectors
2
Neutrophil ELA-2 Secretion Assay
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The ELA-2 secretion by fMLP-cytochalasin B-stimulated neutrophils was determined using N-succinyl-alanine-alanine-valine p-nitroanilide (SAAVNA) as a substrate according to the method of Piwowarski and Kiss [19 (link)] with some modifications. Briefly, 200 µL of the neutrophil suspension (4.0 × 106/mL) in HBSS was pre-incubated in 96-well plates for 15 min at 37 °C with 5% CO2, with the presence or absence of the tested extracts (50 µL) dissolved in HBSS, and tested at a final concentration of 25–150 µg/mL. The cell culture was then stimulated with 50 µL of fMLP (5.6 µg/mL) and cytochalasin B (2.8 µg/mL) for 15 min. After incubation, the plates were stored for 3 min on ice, and then centrifuged (2000 RPM; 10 min; 4 °C). The assay protocol began by adding 50 µL of SAAVNA solution (1.9 mg/mL) to 100 µL of the immediately harvested supernatants in a new 96-well plate. The extent of the released p-nitrophenol was measured at 412 nm over a period of 300 min with 20 min intervals using a microplate reader (Synergy 4). The percentage of the ELA-2 release was calculated in comparison to the control without the investigated extracts. QU (25–75 µM) was used as a positive control. All reagents and media were purchased from Sigma-Aldrich (Seelze).
3
MTT Assay for Cell Viability
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Cell viability
was determined
by MTT assay as previously described with modifications.12 (link) After treatment, 0.5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) was added to the cell culture.
After incubation at 37 °C for 4 h, the formed formazan crystals
were collected and dissolved in dimethyl sulfoxide (DMSO). The absorbance
at 540 nm was read using the Synergy-4 microplate reader.
was determined
by MTT assay as previously described with modifications.12 (link) After treatment, 0.5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) was added to the cell culture.
After incubation at 37 °C for 4 h, the formed formazan crystals
were collected and dissolved in dimethyl sulfoxide (DMSO). The absorbance
at 540 nm was read using the Synergy-4 microplate reader.
4
Quantifying Osteoblast Mineralization
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Cell layers were washed with PBS and extracted as detailed above. ALP activity was determined in the extracts by determining the release of p-nitrophenol from p-nitrophenylphosphate at 37 °C and pH 10.5. The data were normalized to the total protein amount in cell layers.
The degree of mineralization of cell layers was determined using Alizarin Red staining. Briefly, cells were fixed with ethanol and stained with 40 mM Alizarin Red in deionized water at pH 4.2. The bound stain was eluted with 10% (w/v) cetylpyridinium chloride and the absorbance at 562 nm was measured using a Synergy4 multi-mode microplate reader.
The degree of mineralization of cell layers was determined using Alizarin Red staining. Briefly, cells were fixed with ethanol and stained with 40 mM Alizarin Red in deionized water at pH 4.2. The bound stain was eluted with 10% (w/v) cetylpyridinium chloride and the absorbance at 562 nm was measured using a Synergy4 multi-mode microplate reader.
5
Cell Proliferation Assay with Alisertib
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MDA-MB-231/alisertib cells receiving si-NC or si-TCP1 treatment were seeded in 96-well plates. CCK-8 solution (RiboBio, Guangzhou, China) measuring 10 μl was added per well at a specified time (0, 24, 48 h, etc.) based on the protocol. We used a microplate reading element (Synergy4, USA) to measure the cell absorbance at 450 nm.
6
ROS Levels in DLBCL Cells with ZnO NPs
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ROS levels in the DLBCL cells with and without ZnO NPs treatment was measured using DCFH-DA (2ʹ,7ʹ-dichlorodihydrofluorescein diacetate). Suspensions of DLBCL cells (3×105 cells/mL) in culture medium (RPMI 1640 supplemented with 10% FBS) were cultivated in a 96-well culture plate (200 μL/well) containing different concentrations of ZnO NPs (0 μg/mL, 30 μg/mL, 40 μg/mL, 50 μg/mL) for 24 hours at 37 °C with the presence of CO2. Post incubation, the cells were washed twice with PBS and were treated with the DCFH-DA (20 μM) and incubated as before for 1 hour. Next, the dichlorofluorescein (DCF) intensity was measured using microplate reader (Synergy-4) with an excitation wavelength = 338nm and emission wavelength = 500nm. The calculation of DCF intensity in the ZnO NPs treated cells was relative to that of the control cells (ZnO NPs = 0 μg/mL); and the final results were expressed in percentage. The experiments were conducted three times independently.
7
Evaluating Phototoxicity and Dark Toxicity of PDA-FA-Pc Nanomedicine
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The methyl thiazolyltetrazolium (MTT) assays were performed to investigate the in vitro phototoxicity and dark toxicity of PDA-FA-Pc nanomedicine for Hela, MCF-7, HELF and L02 cells. Briefly, cells were seeded into 96-well culture plates at a density of 1×104/well. Cells were attached after 16 hrs. All cells adhered to the bottom after 16 hrs and followed by incubation with PDA-FA-Pc nanomedicine at different concentrations (0.15, 0.3, 0.6, 1.2 and 2.4 mg/mL) for 2 hrs. Unbound PDA-FA-Pc nanomedicine was removed by washing in PBS thoroughly. Then, the cells of phototoxicity groups were followed by 2-min illumination at a dose of 5 J/cm2 via a planar LED light source (680 nm) and incubation of 24 hrs. Next day, cells in each well were mixed with 100 μL of 0.5 mg/mL MTT solution in cell culture medium and incubated for 4 hrs. The formed insoluble purple formazan product was then dissolved in DMSO (100 μL each well). The absorbance of the solution was quantified by measuring at 570 nm with a Synergy 4 multi-mode microplate reader. Meanwhile, dark toxicity experiments of PDA-FA-Pc nanomedicine for all cells were also investigated without illumination.
8
Curcumin Photodegradation Kinetics
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Add 10 mL of curcumin working solution (6.25 μg mL−1) to the beaker, wrap the beaker with tin foil, take 2 mL of curcumin working solution to measure the wavelength of 425 nm with a Synergy 4 multi-mode microplate reader and record it as the ultraviolet absorption value of 0 min. After measurement, pour it back into the beaker, leave it in the dark for 0.75 h, shake well. Take 2 mL of curcumin to measure the UV absorption value of 425 nm wavelength, and take photos of the sample cup for color comparison. After measurement, pour it back into the beaker and stand it for 10 minutes under dark conditions. Repeat the above steps to measure UV absorption at 425 nm wavelengths for 1.5, 3, 6, 12, and 24 h in dark conditions. With the light time as the horizontal coordinate and the photodegradation rate as the vertical coordinate, the line chart of the photodegradation rate of curcumin under dark conditions was drawn, and the color comparison analysis was carried out for each group of sample cups. Photodegradation rate = ((absorption measured at 0 min – absorption measured at N min))/(absorption measured at 0 min).
9
Quantifying β-Arrestin2 Recruitment to MOR
10
Ubiquitination Assay with UbiFlu and Rsp5
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Example 11
During the development of embodiments of the technology provided herein, experiments were conducted to assess the used of the UbiFlu probe with Rsp5. In particular, Ub-Flu (6.0 μM) was mixed with ΔWW Rsp5 (1.0 μM) in 20 mM HEPES 7.5, 50 mM NaCl and then immediately added to a 384 well plate in triplicate (3×70 μL). Fluorescence polarization was observed with the Synergy 4 plate reader every 90 seconds. Polarization units were converted to pmol Ub-Flu. An aliquot of the same reaction mixture was quenched every 15 min for Coomassie and fluorescence gels.
The data collected showed a decrease in fluorescence polarization over the course of approximately 60 minutes, indicating a decrease in the amount of Ub-Flu in the reaction (
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