After stimulation with the tumor-lysate-pulsed DCs, the tumor-antigen-primed T cells were additionally stimulated by incubating with anti-EcOX40 fusion antibodies for 24 h. The calcein-release assay was used for determining the ovarian tumor cell lysis caused by the fusion-antibody-stimulated tumor-antigen-primed T cells in comparison with the tumor-antigen-primed T cells without fusion antibody stimulation (control tumor antigen primed-T cells). Ovarian cancer cells were labeled with 1 μM of calcein-AM (Invitrogen), and 1 × 104 labeled cells were seeded into individual wells of a black 96-wellplate for 12 h. The fusion-antibody-stimulated tumor-antigen-primed T cells and control cells were added separately to the calcein-labeled ovarian cancer cells at target/effector (T:E) ratios of 1:5, 1:10, and 1:20. The cells of all treatments were cultured at 37 °C in CO2 incubator for 24 h. After incubation, 100 μL of the supernatant from each well (triplicate wells for each treatment) was collected, and the fluorescence intensity of the released calcein was measured using the BioTek Synergy H1 Multimode Reader (BioTek). Two independent and reproducible experiments were performed using T cells of two blood donors whose MHC class-I partially matched that of the ovarian cancer cells. Specific lysis of the cancer cells was calculated as follows:
Biotek synergy h1 multimode reader
Manufactured by Agilent Technologies
Sourced in United States
The BioTek Synergy H1 Multimode Reader is a versatile laboratory instrument designed for various detection modes, including absorbance, fluorescence, and luminescence. It is capable of performing a wide range of assays and provides accurate and reliable data.
Automatically generated - may contain errors
Lab products found in correlation
Calcein am, by Thermo Fisher Scientific (1 mentions)
Bhb colorimetric assay kit, by Cayman Chemical (1 mentions)
Antioxidant assay kit, by Cayman Chemical (1 mentions)
Ursolic acid, by Merck Group (1 mentions)
Mjfr 14 6 4 2, by Abcam (1 mentions)
96 well plate, by Corning (1 mentions)
Meropenem, by Agilent Technologies (1 mentions)
Methanolic dpph solution, by Merck Group (1 mentions)
7 protocols using biotek synergy h1 multimode reader
1
Fusion Antibody-Mediated Cytotoxicity Assay
2
Antioxidant and Metabolite Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Commercial colorimetric assay kits were used to determine muscle/liver/brain total antioxidant capacity (Antioxidant assay kit #709001; Cayman Chemical, Ann Arbor, MI, USA) and serum BHB levels (BHB colorimetric assay kit #700190; Cayman Chemical), respectively, according to manufacturer’s instructions. For total antioxidant capacity determination, approximately 100 mg of frozen muscle/brain/liver was homogenized in kit assay buffer and centrifuged according to manufacturer’s instructions, and supernatants were assayed. Given that brain tissue in 8 month-fed rats was devoted to mitochondrial assays and Western blotting, brain total antioxidant capacity determination was not performed because of lack of available tissue. Following assay execution, all plates were read in a UV-Vis microplate reader (BioTek Synergy H1 Multi-Mode Reader; BioTek, Winooski, VT, USA) at absorbance readings according to manufacturer’s recommendations.
3
Quantifying Total Flavonoid Content
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To determine the amount of total flavonoid content (TFC) in a 96-well microplate [51 (link)], an aliquot (30 μL) of the methanolic extract solution was mixed with 120 μL of sterile distilled water and 10 μL of 5 % (w/v) NaNO2 solution. The mixture was incubated for 5 min at 25 °C before the addition of 10 % (w/v) AlCl3 solution (10 μL). The mixture was allowed to stand for 6 min at ambient temperature before the addition of 1 N NaOH solution (60 μL). The reaction mixture was then diluted to a volume of 300 μL with sterile distilled water, thoroughly mixed, and incubated at 25 °C for 15 min. The intensity of the pink color was measured at 510 nm using a microplate reader (BioTek Synergy H1 Multimode Reader, Agilent Technologies, USA). To create the standard curve, quercetin (Fisher Scientific, USA, purity >95 %) was used in concentrations ranging from 0 to 100 μg/mL. The results were expressed as mg quercetin equivalent per gram of dry weight (mg QCE/g), and sterile distilled water was used as the negative control.
4
Quantifying Total Triterpenoid Content
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The total triterpenoid content (TTC) was determined using a modified method of Wei et al. (2015) [50 (link)] and reported as mg ursolic acid (Sigma-Aldrich, USA, purity >90 %) equivalent per gram of dry weight. Initially, the extracts were diluted ten times, and 30 μL of the resulting solution was placed into a 96-well microplate. The extract was evaporated using a hot air oven, followed by the addition of 10 μL of fresh mixed 5 % (w/v) vanillin-acetic solution (vanillin purchased from Sigma-Aldrich, USA, purity 99 %) and 38 μL of 98 % (v/v) sulfuric acid. After mixing, the solution was incubated at 70 °C for 30 min and cooled before being diluted to 200 μL with acetic acid. The absorbance of the resulting solution was measured at 573 nm using a microplate reader (BioTek Synergy H1 Multimode Reader, Agilent Technologies, USA) against a blank that contained all reagents and solvents except for the sample solution. To create the standard curve, ursolic acid (Sigma-Aldrich, USA, purity ≥90 %) was used in concentrations ranging from 0 to 100 μg/mL. The results were expressed as mg ursolic acid equivalent per gram of dry weight (mg UAE/g), and sterile distilled water was used as the negative control.
5
Quantification of α-Synuclein Aggregates
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For the measurement of α-synuclein aggregates, 96-well plates (Corning Costar, Glendale, AZ, USA) were coated overnight at 25 °C with 1 μg/mL of the mouse monoclonal antibody Syn-F2 (50 μL/well) in 100 mM NaHCO3 (pH 9.6). The plates were washed three times with wash buffer (50 mM Tris pH 7.4, 150 mM NaCl, 0.1% Tween-20). Standards and samples were diluted in 10 mM Tris-Cl, pH 7.6, 100 mM NaCl, 0.1% Tween-20 and 1% BSA (TBS-T/BSA buffer) and incubated for 2.5 h at 37 °C. After washing three times, the rabbit monoclonal antibody MJFR-14-6-4-2 (Abcam, Cambridge, UK) was added at a concentration of 74 ng/mL and incubated for 1 h at 25 °C. The wells were washed again three times, and the anti-rabbit IgG-HRP antibody (Agilent, Santa Clara, CA, USA) was applied at a concentration of 16.7 ng/mL (1:15,000 dilution in TBS-T/BSA buffer) for 30 min at 4 °C. The bound HRP was detected by adding 50 μL/well of HRP substrate (Luminata Crescendo ELISA HRP chemiluminescent substrate, Merck Millipore), and the chemiluminescence in relative light units was measured after 5 min in a Biotek Synergy H1 multimode reader (Agilent, Santa Clara, CA, USA).
6
Multidrug-Resistant Klebsiella pneumoniae Characterization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The Klebsiella pneumoniae strain HCD1 (KpHCD1) was isolated from the urine culture of a patient at the Hospital Copa D’Or in Rio de Janeiro, Brazil. The antibiogram indicated resistance to amikacin, amoxicillin, ampicillin, cephalothin, cefepime, ceftriaxone, cefuroxime, Ciprofloxacin, ertapenem, meropenem, nitrofurantoin, norfloxacin, piperacillin, polymyxin B, and sulfamethoxazole, classifying the HCD1 strain as XDR [5 (link)]. Minimum inhibitory concentrations (MICs) of meropenem, amikacin and polymyxin B (all from Sigma-Aldrich Brazil, Sao Paulo, SP, Brazil) were obtained according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI), based on broth microdilution in cation-adjusted Mueller–Hinton II broth (BD DIFCO, Lakes, NJ, USA) in 96-well polypropylene microtiter plates. From the MICs, growth curves in the presence of meropenem, amikacin, polymyxin B, and a combination of these antibiotics were performed in triplicate; growth kinetics were recorded every hour for 24 h at 37 °C in a microplate reader (BioTek Synergy H1 Multimode Reader, Agilent, CA, USA).
7
Evaluation of DPPH Radical Scavenging Activity
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To evaluate the scavenging activity of DPPH free radicals, Govarthanan et al. (2015) [51 (link)] method was used with a slight modification. Initially, a methanolic extract of the sample (diluted 1:10) or gallic acid at various concentrations (ranging from 6.25 to 200 μg/mL) was mixed with 100 μL of 0.1 mM methanolic DPPH solution (Sigma-Aldrich, USA), followed by incubation for 20 min at room temperature in the dark. The resulting mixture was then measured at a wavelength of 517 nm using a microplate reader (BioTek Synergy H1 Multimode Reader, Agilent Technologies, USA). The negative control was sterile distilled water. The scavenging activity of the extracts was determined using the following formula: inhibition (%) = [(absorbance of control – absorbance of sample) x 100]/absorbance of control, where absorbance control is the absorbance of DPPH solution without extract and Abs sample is the absorbance of the sample with DPPH solution.
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!