Caspase 3 activity was measured using the Caspase-Glo® 3/7 (Promega Co., Madison, WI), in which 50,000 cells/well were initially seeded in a flat bottom 96-well plate. Cells were incubated with equal volumes of the reagent to the culture medium for 1 h, and a Varioskan Flash Reader (Thermo Fisher Scientific, Waltham, MA) was used to assess relative luminescence. Caspase 9 activity was measured using the Caspase-9 Fluorometric assay (R&D Systems, Inc. Minneapolis, MN). Each sample contained the cell lysate (100 μg of protein in 50 μL), 50 μL of 2X reaction buffer, and 5 μL of caspase 9 fluorometric substrate (LEHD-AFC). The reaction was incubated for 1 h at 37°C in a flat bottom 96-well microplate. Fluorescence signal was indicative of caspase activation, and it was measured on a fluorescent plate reader (Gemini SpectraMax, Molecular Devices, CA, USA).
Spectramax gemini
Manufactured by Molecular Devices
Sourced in United States, Germany
The SpectraMax Gemini is a multi-mode microplate reader designed for a variety of applications in life science research. It features optical components for absorbance, fluorescence, and luminescence detection, enabling users to perform diverse assays on a single platform.
Automatically generated - may contain errors
Lab products found in correlation
Hemocytometer, by Marienfeld (4 mentions)
Calcein am, by Thermo Fisher Scientific (4 mentions)
96 well plate, by Greiner (4 mentions)
Atp colorimetric fluorometric assay kit, by Merck Group (3 mentions)
Fuji las 3000 system, by Fujifilm (3 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Softmax pro software, by Molecular Devices (2 mentions)
Caspase glo 3 7 assay, by Promega (2 mentions)
Quant it picogreen dsdna assay kit, by Thermo Fisher Scientific (2 mentions)
Bca protein assay, by Thermo Fisher Scientific (2 mentions)
Varioskan flash reader, by Thermo Fisher Scientific (1 mentions)
Caspase glo 3 7, by Promega (1 mentions)
7 amc, by Merck Group (1 mentions)
Alamarblue reagent, by Thermo Fisher Scientific (1 mentions)
Cyquant cell proliferation assay kit, by Thermo Fisher Scientific (1 mentions)
Human umbilical vein endothelial cells (huvec), by Lonza (1 mentions)
Egm 2 bulletkit, by Lonza (1 mentions)
Eclipse ts100, by Nikon (1 mentions)
Benzoyl arginine p nitroanilide bapna, by Merck Group (1 mentions)
2 7 dichlorofluorescin diacetate, by Thermo Fisher Scientific (1 mentions)
134 protocols using spectramax gemini
1
Caspase 3 and 9 Activity Assays
2
ROS Quantification in Tissue Homogenates
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The ROS activity was estimated using an ELISA kit in accordance with manufacturer instructions of sigma aldrish as described previously [66 (link)]. Briefly, the fresh tissue homogenate was centrifuged for 20 min at 3000RPM, collected, and analyzed for ROS levels. The relative fluorescence unit (RFU) was measured after 30 min by using Gemini Spectra MAX microplate reader (Molecular Devices, Sunnyvale, CA [67 (link)].
3
Fluorometric Assay of Soil Enzyme Activities
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The substrates 7-amino-4-methyl coumarin hydrochloride (7-AMC-leucine), which was used to measure LAP activity, and 4-methylumbelliferyl-β-D -glucosaminidine (4-MUB-NAG), which was used to measure NAG, as well as their respective fluorogenic compounds (4-MUB and 7-AMC) were obtained in crystalline form from Sigma–Aldrich. For each treatment, the activities were assayed in six replicates using black microplates by adding 50 μL of the 200 μM substrate solution and 200 μL of the litter extract diluted in acetate buffer. This solution was incubated at room temperature for 3 h for the LAP assays and for 30 min for the NAG assays at pH 6. The reactions were terminated by adding between 8 and 20 μL of 1 M sodium hydroxide solution to raise the pH to 9 and optimize the fluorescence (Bell et al., 2013 (link)). After excitation at 365 nm, the fluorescence emission intensity of the sample was measured at 460 nm using a microplate fluorimeter (Spectramax Gemini, Molecular Devices Corporation, Sunnyvale, CA, USA) and corrected for quenching as described in German et al. (2011) (link). We determined the LAP activity by referencing a standard curve obtained from commercial LAP from porcine kidney (Sigma–Aldrich). A standard curve of the fluorescence intensities for the specific fluorogenic product (4-MUB) of the substrate (4-MUB-NAG) was used to quantify the NAG activity.
4
Quantifying Cell Viability After PFT Exposure
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For the viability experiments, 2 × 106 cells were washed in Ca2+ Tyrode’s buffer and incubated with PFTs as indicated in Ca2+ Tyrode’s buffer (final volume 1 ml) for 30 min at room temperature (aerolysin was activated with trypsin prior). In all cell survival experiments, cells were pelleted after stimulation with PFTs and grown in 3 ml RPMI 1640 medium with penicillin/streptomycin and 10% FBS for 24 h. For measuring viability, 0.1 volume of alamar Blue reagent (Thermo Fisher Scientific, Waltham, MA, USA) was pipetted directly into 100 μL aliquots of the cells in culture medium and incubated for 3–4 h at 37 °C under protection from direct light. Fluorescence emission was measured with a microplate reader (SpectraMax Gemini; Molecular Devices, San Jose, CA, USA) at 590 nm with an excitation wavelength of 560 nm.
5
Calibrated Automated Thrombography of FVIII
6
Fluorimetric Assay for Cathepsin L Activity
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
CatL activity was measured using fluorimetric CatL activity assay kit (BioVision, USA) according to the manufacturer's instructions. Cells were lysed in lysis buffer. Cell lysate (50 μl) was incubated with 50 μl reaction buffer containing 1 μmol/L Ca074 (a CatB enzymatic activity-specific inhibitor, Calbiochem, USA) for 15 min to irreversibly inhibit CatB.[18 (link)] CatL substrate (Ac-Phe-Arg- amino-4-trifluoromethylcoumarin [AFC], 200 μmol/L final concentration) was then added and the mixture was incubated for 1 h at 37°C. Release of free AFC was measured at λex 400 nm and λem 505 nm using a fluorescence plate reader SpectraMax Gemini (Molecular Devices, USA). Fold increases in CatL activity were determined by comparing the relative fluorescence units against the levels of the controls.
7
Protein Unfolding Dynamics in GndHCl
8
Evaluating ASC-Sheets on Endothelial Cell Proliferation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To test the effect of ASC-sheets on endothelial cell proliferation, human umbilical vein endothelial cells (HUVEC, Lonza) at P4 were seeded at a density of 5000 cells/cm2 in a 96-wells plate and in a 24-wells plate and cultured overnight in endothelial growth medium (EGM-2 bullet kit, Lonza). The next day, cells were starved with 0.5% FBS in LG-DMEM for 3 h. Then, HUVEC were refreshed with either control medium (LG-DMEM 1% FBS) mixed with EGM medium (1:1) or medium conditioned by ASCs mixed with EGM medium (1:1). After 0, 1, 2, 3, and 4 days endothelial cell proliferation and viable cell numbers were determined with the Cyquant® cell proliferation assay kit (Invitrogen) and MTT assay, respectively. Combining the results from these assays will allow to (indirectly) have an indication about the proliferation. According to the manufacturers’ protocol culture plates at -80οC were frozen after removal of medium. The proliferation on each day was analyzed using known numbers of HUVEC as a DNA standard. At room temperature, 200 μl of CyQuant GR dye/lysis buffer was added to each well and incubated 5 min before reading the plate with the fluorescence microplate reader SpectraMax Gemini (Molecular Devices)
The MTT assay was based on the Mossman’s protocol [24 (link)] to check for metabolically active cells.
The MTT assay was based on the Mossman’s protocol [24 (link)] to check for metabolically active cells.
9
Intracellular ROS Quantification by DCFH-DA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The generation of ROS in cells was evaluated by a fluorometry assay using intracellular oxidation of nonfluorescent probe 2, 7‐dichlorofluorescein diacetate (DCFH‐DA). DCFH‐DA can passively diffuse into cells and be deacetylated by esterase to form nonfluorescent 2,7‐dichlorofluorescein (DCFH). In the presence of ROS, DCFH reacts with ROS to form the fluorescent product DCF, which is trapped inside the cells. When the membrane is oxidized and damaged, the fluorescence will attenuate significantly. PANC‐1 and BxPC‐3 cells were incubated with corresponding treatment in a 6‐well plate with 3 × 105 cells/well; 30 min before the end of treatment, the cells were incubated with DCFH‐DA at 37 °C for 20 min. After treatment as mentioned above, DCF fluorescence intensity was detected by fluorescence spectrometry (Spectramax Gemini, Molecular Devices, San Jose, CA, USA) with an excitation wavelength of 490 nm and an emission wavelength of 530 nm. The results were expressed as relative fluorescence intensity per 104 cells.
10
Deacetylase Activity Assay for SIRT1
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!