The cytotoxic activity of GPA7-28z T cells and mock-transduced T cells was evaluated in a Calcein-AM release assay17 (link). In brief, target cells including peptide-pulsed T2 cells and melanoma cells were labeled with 15 μM calcein-AM for 30 min at 37°C. T2 cells were pre-incubated with a series for ten-folded diluted peptide (10−5 ~ 103 nmol/mL) for 3 h at 37°C. After washes in complete medium twice, target cells were adjusted to 105/ml and 100 μL added per well of a 96-well V-bottom plate. Subsequently, effector cells were added to wells at the given ratios. The test was performed in four replicates for each E/T ratio, with at least six replicate wells for spontaneous (only target cells in complete medium) and maximum release (only target cells in medium plus 2% Triton X-100). After incubated at 37°C for 4 hours, supernatants were harvested and transferred into a black 96-well plate. Fluorescence was measured using a Synergy TM H4 Multi-Mode Microplate Reader (BioTek instruments, Winooski, VT) (excitation filter: 485 ± 9 nm; band-pass filter: 530 ± 9 nm). Percent specific lysis was calculated as (experimental release - spontaneous release)/(maximum release – spontaneous release) × 100. For antibody blocking assay, gp100-pulsed T2 cells were pre-incubated with 10 μg/mL mouse anti-HLA-A2 antibody BB7.2 for 30 min before mixing with GPA7-28z T cells.
Synergy h4 multi mode microplate reader
Manufactured by Agilent Technologies
Sourced in United States
The Synergy H4 Multi-Mode Microplate Reader is a versatile laboratory instrument designed for a wide range of applications. It is capable of detecting multiple detection modes, including absorbance, fluorescence, and luminescence, within a single microplate.
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Lab products found in correlation
Dmem f12 medium, by Thermo Fisher Scientific (3 mentions)
Cellstar μclear 96 well tissue culture plates, by Greiner (3 mentions)
Dnase 2, by Merck Group (2 mentions)
Celltiter 96 aqueous non radioactive cell proliferation assay, by Promega (2 mentions)
Lipofectamine ltx, by Thermo Fisher Scientific (1 mentions)
Prl tk plasmid, by Promega (1 mentions)
Luc pair duo luciferase assay kit 2, by GeneCopoeia (1 mentions)
Gelatin from porcine skin, by Merck Group (1 mentions)
Ix53 inverted fluorescent microscope, by Olympus (1 mentions)
Tryple, by Thermo Fisher Scientific (1 mentions)
384 pin tool device, by V&P Scientific (1 mentions)
Fluorescence microscope, by Leica (1 mentions)
Mitosox, by Thermo Fisher Scientific (1 mentions)
Dcfh da, by Merck Group (1 mentions)
Mitosox, by Agilent Technologies (1 mentions)
Originpro 8, by OriginLab (1 mentions)
Caspase glo 3 7 kit, by Promega (1 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
Trichloroacetic acid, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole dihydrochloride dapi, by Thermo Fisher Scientific (1 mentions)
24 protocols using synergy h4 multi mode microplate reader
1
Cytotoxicity Assay for GPA7-28z T Cells
2
Transcriptional Regulation of Smooth Muscle α-Actin
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C2C12 cells were plated on 12‐well dishes coated with 0.1% gelatin from porcine skin (Sigma‐Aldrich) and transiently transfected using Lipofectamine LTX (Thermo Fisher Scientific) according to the manufacturer's instructions, resulting in a transfection efficiency of approximately 70%. Cells were cotransfected with pxj40‐HA expression plasmids encoding HA‐tagged full‐length MYPN or PALLD isoform 4 (25, 50, and 100 ng) and/or FLAG‐tagged MRTF‐A (3 ng), as indicated, a pGL3 basic rat smooth muscle α‐actin promoter firefly luciferase reporter plasmid26 (250 ng), and the pRL‐TK plasmid (Promega) expressing Renilla luciferase under the control of the thymidine kinase promoter to normalize for transfection efficiency (5 ng). C2C12 myoblasts were induced to differentiate 24 h after transfection and were lysed after 48 h of differentiation. Firefly and Renilla luciferase activities were measured on a SynergyTM H4 Multi‐Mode Microplate Reader (BioTek) using the Luc‐Pair Duo‐Luciferase Assay Kit 2.0 (GeneCopoeia) according to the manufacturer's instructions. Firefly luciferase activity was normalized to Renilla luciferase activity to account for variations in transfection efficiency. All experiments were performed in triplicate and repeated at least three times.
3
ROS Quantification in HUVECs
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A dichloro-dihydro-fluorescein diacetate (DCFH-DA) detection kit was used to assess the ROS level in HUVECs. After pretreatment with the 5 µg/mL of Mv, Mv-3-glc, Mv-3-gal, or BAE for 24 h and continuing with 5.5 mM or 30 mM glucose for 24 h, cells were washed with PBS, and then 10 µM DCFH-DA was added to each well and reacted for 20 min at 37 °C, and the cells were washed thoroughly with PBS. A group of cells was visualized under an IX53 inverted fluorescent microscope (Olympus, Tokyo, Japan) with 530 nm emission and 485 nm excitation filters immediately. All images presented are in ×200 magnification. Another group of cells was collected after dissociation, and fluorescence was recorded by a Synergy H4 multi-mode microplate reader (BioTek Instruments Inc., Winooski, VT, USA). The total fluorescence intensity of cells in each well was noted, and ROS generation was measured as a fold of the control.
4
Sulforhodamine B Cytotoxicity Assay
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An assay was performed after 72 h of exposure to varying concentrations of the tested compounds (from 0.1 to 100 µg/mL); the time of incubation was chosen according to studies of Yong et al. [38 (link)] who analyzed the various effects of xanthohumol in 24, 48 and 72 h incubation periods and find out that the majority of xanthohumol-induced effects occurred significantly in the 72 h treatment period. The cells were fixed in situ by gently adding cold 50% trichloroacetic acid (50 µL per well). The plates were incubated at 4 °C for 1h and then washed five times with tap water. Next, the cellular material fixed with TCA was stained by addition to each well 50 µL of 0.4% sulforhodamine B dissolved in 1% acetic acid and incubated for 30 min at room temperature. Unbound dye was removed by washing the plates five times with 1% acetic acid, whereas the protein-bound dye was extracted with 150 µL of 10 mM unbuffered Tris base for determination of the optical density (λ = 540 nm) in Synergy H4 multi-mode microplate reader (Bio Tek Instruments, Winooski, VT, USA).
5
Bioluminescence Assay for FGF14-Nav1.6 Interaction
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HEK293 cells stable expressing CLuc-FGF14 and CD4-Nav1.6-NLuc (Clone-V) were grown for 24–48 h. Clone-V cells were detached using TrypLE (Gibco, Waltman, MA, United States), triturated in medium, and seeded in white, clear-bottom CELLSTAR μClear® 96-well tissue culture plates (Greiner Bio-One) at ∼0.8 × 105 cells per well in 200 μl of medium. The cells were treated for 12 h in a growth medium supplemented with 100 μl of serum-free, phenol red–free DMEM/F12 medium (Invitrogen) containing PLEV or EYYV (1–250 μM). The final concentration of DMSO was maintained at 0.5% for all wells. Following 12 h incubation at 37°C, the luminescence reaction was initiated by injection of 100 μl substrate solution containing 1.5 mg/ml of D-luciferin dissolved in PBS (final concentration = 0.75 mg/ml) by the Synergy™ H4 Multi-Mode Microplate Reader (BioTek). LCA readings were performed at 2 min intervals for 20–30 min, integration time 0.5 s, while cells were maintained at 37°C throughout the measurements. Detailed LCA method can be found in previous studies (Shavkunov et al., 2012 (link); Ali et al., 2014 (link), 2016 (link), 2018 (link); Hsu et al., 2015 (link); Wadsworth et al., 2019 (link), 2020 (link); Singh et al., 2020 (link)).
6
Fluorescence-Based Meprin Enzyme Assay
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Both assays followed the same general protocol53 (link). 5 µL of 2x enzyme solution (2.6 and 0.1 nM for meprin α and β, respectively) in assay buffer (50 mM Hepes, 0.01% Brij-35, pH 7.5) were added to solid bottom black 384 low volume plates (Nunc, cat# 264705). Next, 75 nL of test compounds or pharmacological control (actinonin) were added to corresponding wells using a 384 pin tool device (V&P Scientific, San Diego). After 30 min incubation at RT, the reactions were started by addition of 5 µL of 2x solutions of substrates (20 µM, mepin α Mca-YVADAPK-K(Dnp); and for meprin β Mca-EDEDED-K(Dnp). Reactions were incubated at RT for 1 h, after which the fluorescence was measured using the Synergy H4 multimode microplate reader (Biotek Instruments) (λexcitation = 324 nm, λemission = 390 nm).
Three parameters were calculated on a per-plate basis: (a) the signal-to-background ratio (S/B); (b) the coefficient for variation [CV; CV = (standard deviation/mean) × 100)] for all compound test wells; and (c) the Z- or Z′-factor55 (link). Z takes into account the effect of test compounds on the assay window, while Z′ is based on controls.
Three parameters were calculated on a per-plate basis: (a) the signal-to-background ratio (S/B); (b) the coefficient for variation [CV; CV = (standard deviation/mean) × 100)] for all compound test wells; and (c) the Z- or Z′-factor55 (link). Z takes into account the effect of test compounds on the assay window, while Z′ is based on controls.
7
Quantification of Cellular ROS Levels
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Both cytosolic and mitochondrial ROS were measured with 2′,7′-dichlorodihydrofluorescein-diacetate (DCFH-DA, at a wavelength of 525 nm, Sigma-Aldrich, USA) and MitoSOX (a probe of mitochondrial ROS, at a wavelength of 580 nm, Invitrogen, USA), respectively. After incubation with 10 μM DCFH-DA for 30 min or 5 μM MitoSOX for 15 min in a dark room, treated INS-1 cells were washed twice with RPMI 1640 containing no FBS and immediately analyzed to evaluate the levels of ROS with either a Synergy H4 Multi-Mode Microplate Reader (Biotek, USA) or fluorescence microscope (Leica, Germany).
8
DCFH-DA Assay for ROS Quantification
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Dichloro-dihydro-fluorescein diacetate (DCFH-DA) detection kit was used to assess the ROS level in HRCECs. Briefly, the cells were seeded in 96-well plates, treated with different samples to incubate for 24 h and cultured with high glucose (30 mmol/L) for 24 and 48 h. After washing cells with phosphate-buffered saline (PBS), 10 μmol/L DCFH-DA was added to each well and reacted for 20 min at 37°C. The cells were collected after dissociation, and fluorescence was recorded by a Synergy H4 Multi-Mode Microplate Reader (BioTek Instruments, Inc., Winooski, VT, USA) with 488-P excitation and 525-P emission filters. The total fluorescence intensity of cells in each well was noted, and ROS generation was measured as fold of the control (each NG group, 24 and 48 h).
9
MTT Assay for Cell Viability
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The cell viability was determined by MTT method. The cells were cultured with high glucose (30 mmol/L) for 24 and 48 h with or without BAE, Mv, Mv-glc, or Mv-gal pretreatment for 24 h. Ten microliters of MTT (5 mg/mL) was added to each well and cultured for 4 h. After the removal of MTT solution, cell crystal was dissolved by adding 100 μL DMSO (dimethylsulfoxide) and shaking 10 min slowly. The absorbance was measured at 550 nm on a Synergy H4 Multi-Mode Microplate Reader (BioTek Instruments, Inc., Winooski, VT, USA). The reader was controlled via Hyper Terminal Applet ELISA software. The cells were cultured only when normal glucose (5.5 mmol/L) was used as the control group. The blank group used the wells without cells. The cell viability was determined with the following formula: Cell viability(%) = (sample group OD value − blank group OD value)/(control group OD value − blank group OD value) × 100%.
10
Shh-N Pathway Activation Assay
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Shh-N conditioned medium was prepared as previously described (Chen et al. 2002b (link)). Cells were cultured, transfected, and the Hh pathway responsive reporter cell lines were constructed according to methods provided in supplementary data. Monoclonal NIH3T3 (ATCC, Manassas, VA) cells stably transfected with reporter gene were separately plated at 1 × 105 cells per well of 24-well plates. After cells reached confluence in about 2 days, they were shifted to serum-free medium containing 20% Shh-N conditioned medium or 20% HEK293 conditioned medium treatments. After 36 h, cells were lysed and luciferase activity was assayed on a Synergy-H4 Multi-Mode Microplate Reader (Bio-Tek Instruments, Winooski, VT) according to the protocol in Molecular Cloning (Sambrook and Russell 2001 ). Dozens of monoclonal cell lines were analyzed, and a cell line with optimal sensitivity to Shh-N stimulation and normal morphology was designated as NIH3T3-12Gli cell line.
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