To assess overall cellular health after morphine treatment SH-SY5Y cells were plated at 7.5 × 104 cells per well in a clear 96-well plate coated with poly-D -lysine (354210, Corning®, Corning, NY, United States) for 3 days in complete media or in complete media with 5–1,500 μM morphine in a humidified incubator at 37°C with 5% CO2. After 3 days, media was removed and replaced with 100 μL pre-warmed complete media along with 10 μL of a 5 mg/mL thiazolyl blue tetrazolium bromide (00697, Chem-Impex International, Inc., Wood Dale, IN, United States) solution in 1X phosphate buffered saline and incubated 4 h (van Meerloo et al., 2011 (link)). Next, 100 μL of a 0.01 N HCl solution with 10% w/v sodium dodecyl sulfate was added. Plates were wrapped in parafilm and incubated overnight. Absorbance was read at 570 nm using a BioTek Synergy 2 multi-well plate reader.
Synergy 2 multi well plate reader
Manufactured by Agilent Technologies
Sourced in United States
The Synergy 2 multi-well plate reader is a versatile instrument designed for various laboratory applications. It can measure absorbance, fluorescence, and luminescence in 6- to 384-well microplates. The Synergy 2 utilizes advanced optical components and detection technologies to provide accurate and reliable results.
Automatically generated - may contain errors
7 protocols using synergy 2 multi well plate reader
1
Assessing Morphine's Impact on SH-SY5Y Cells
2
Quantifying β-galactosidase Activity in E. coli
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Biomass of E. coli strains harboring lacZ reported fusions was collected from the interaction and distant macrocolony zones. Cells were resuspended in Z-buffer and normalized by OD578. β-galactosidase activity was assayed by using onitrophenyl-β-D-galactopyranoside (ONPG) as a substrate. The production of yellow color in each sample was quantified by measuring the absorbance at 415 nm using a Synergy 2 multiwell plate reader (BioTeK). The β-galactosidase activity was reported as μmol of o-nitrophenol per min per mg of cellular protein. The experiments were done at least in quadruplicates. Individual and average data with error bars are shown in the figure.
3
Caco-2 Glucose Uptake Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Caco-2 cells were seeded in 96-well plates at the density of 1 × 104 cells/well. After 48 h of incubation, cells were treated. For glucose uptake studies, Caco-2 cells were placed on 100 μL of glucose-free media for two h containing either 100 μM pure peptides (AKSPLF, ATNPLF, FEELN, LSVSVL), or 100 μM phloretin (PHL), or 10 mg/mL PFRA; then, the media was replaced with 100 μL of glucose-free media containing 100 μM fluorescent d -glucose derivative 2-NBDG. Fluorescence readings were taken after 30, 60 120 and 180 min at 37 °C. Glucose uptake was stopped by washing three times with a two-fold volume of ice-cold PBS. A Synergy2 multi-well plate reader (Biotek, Winooski, VT) at 485 nm excitation and 535 nm emission filter measured fluorescent intensity. The cells were lysed in 30 μL RIPA lysis buffer, and protein concentrations were measured using DC protein assay (Bio-Rad Laboratories, Hercules, CA). The results were expressed as the percentage of glucose uptake relative to the untreated control and normalized to protein concentration.
4
Biofilm Formation in E. coli: Effects of Bacillaene
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To examine the effect of bacillaene or pksL extract on E. coli submerged biofilms the multiwell plate culture system was used. Briefly, overnight cultures were diluted into fresh salt-free LB medium to an initial OD578 of 0.05. One hundred microliter aliquots of these cultures, with or without supplementation of appropriated volumes of purified bacillaene or pksL extract, were loaded into wells of 96-well plates. The plates were sealed with breathe-easy sealing membrane (Sigma) to prevent evaporation and incubated at 28 °C without shaking. After 24 h of incubation, the planktonic cell suspensions were recovered, transferred to new wells and subjected to OD578 measurement with a Synergy 2 multiwell plate reader (BioTeK). In parallel, wells containing (or not) adhered biofilm biomass were washed three times with 200 μl distilled water and biofilms were subsequently stained with 125 μl of 0.1% CV for 15 min at room temperature. Excess CV was removed and the wells were rinsed three times with 200 μl distilled water and left to air dry. CV-stained biofilms on the wells were imaged using a D3100 reflex camera. The bound dye in each well was then solubilized with 125 μl of 30% acetic acid in water. The CV solutions were transferred to new wells and quantified by measuring the absorbance at 550 nm using a Synergy 2 multiwell plate reader.
5
HA Quantification in HaCaT Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The HaCaT cells (3 × 105 cells/well) were added to a 6-well plate for 12 h and starved for more than 6 h in serum-free DMEM to eliminate any FBS effects. Cells were then incubated with different concentrations of LMFAb or the positive control (rhKGF, 20 ng/mL) for 24 h when media were collected and centrifuged sequentially at 500×g, 800×g, and 1000×g for 10 min each. Supernatants (conditioned media; 50 μL/well) were analyzed for HA using an ELISA kit (R&D Systems). HA levels were analyzed using a Synergy 2 multi-well plate reader (Bio-Tek Instruments) at 450 nm.
6
HaCaT Cell Viability Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HaCaT cell viabilities were measured by a water-soluble tetrazolium salt assay using the EZ-CyTox assay kit, as previously described by Kim et al. [23 (link)]. Briefly, HaCaT cells (3 × 103 cells/well) were seeded into 96-well cell culture plates and treated with different concentrations of LMF absolute for 48 h in a humidified atmosphere of 95 % air/5 % CO2. Cells were then incubated with EZ-CyTox reagent (10 μL/well) for 30 min under the same conditions, and viabilities were assessed at 450 nm using a Synergy 2 multi-well plate reader (Bio-Tek Instruments, Winooski, VT, USA). Cell viabilities were expressed as percentages of untreated controls. The results obtained were used to set concentration ranges for other experiments.
7
Cytotoxicity and Proliferation Assays
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cell growth curve experiment provided information on the doubling time of the HUVECs. The ACNs and PAs cytotoxicity assay was conducted so that the optimum concentration and exposure time of the active compounds was used for the rest of the experiments. Different concentrations (0.001 μg/ml -1000 μg/ml for ACNs and 0.001 μg/ml -500 μg/ml for the PAs) and different exposure times were tested (30 min, 1 h, 3 h, 6 h, 12 h, 24 h, 48 h and 72 h). Cell proliferation and cytotoxicity assays were conducted by using the alamarBlue assay (Life Technologies, DAL1025). Cells for both cell proliferation and cytotoxicity assays were measured by using Synergy 2 multiwell plate reader (Bio-Tek Instruments Inc., Winooski, VT) with excitation⁄emission (530 nm-560 nm ⁄ 590 nm) as previously described (Stoddart, 2011) .
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!