The growth inhibition curve of C. tropicalis by ISO was performed following the methodology proposed by [23 (link)], with minor modifications. Isolates of C. tropicalis were cultured in SDB medium for 24 h at 37 °C. The fungal inoculum was standardized until reaching a cell concentration of 106 CFU/mL in glass tubes, then the ISO was added (the MIC for each isolate) and the tubes were incubated at 37 °C for 48 h. Subsequently, 1 mL was taken from each tube at times 0, 2, 4, 8, 12, 24, 36 and 48 h and read at 530 nm in a SYNERGY LX microplate reader (Biotek). Tubes with the fungal inoculum and FLZ were used as controls. All assays were performed in triplicate.
Synergy lx microplate reader
Manufactured by Agilent Technologies
Sourced in United States
The Synergy LX microplate reader is a compact and versatile instrument designed for various plate-based assays. It features a sensitive detection system and can measure absorbance, fluorescence, and luminescence in microplates.
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Lab products found in correlation
Mir 22 3p mimic, by GenePharma (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Dual luciferase reporter assay system, by Promega (1 mentions)
Lipofectamine 2000, by GenePharma (1 mentions)
Prl tk, by Promega (1 mentions)
Tmb substrate, by Cell Signaling Technology (1 mentions)
Pngase f, by New England Biolabs (1 mentions)
Ultra 55 field emission scanning electron microscope, by Zeiss (1 mentions)
Inverted fluorescence microscope, by Olympus (1 mentions)
Ultraflextreme maldi tof tof mass spectrometer, by Bruker (1 mentions)
Autopore 9500, by Micromeritics (1 mentions)
Evos m5000 imaging system, by Thermo Fisher Scientific (1 mentions)
Gen5 data analysis software, by Agilent Technologies (1 mentions)
Prism software, by GraphPad (1 mentions)
E el r0041c, by Elabscience (1 mentions)
Hyp assay kit, by Solarbio (1 mentions)
Hydrogen peroxide assay kit, by Abcam (1 mentions)
9 protocols using synergy lx microplate reader
1
C. tropicalis Growth Inhibition Curve by ISO
2
Evaluating C. tropicalis Viability on ISO
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To evaluate the cell viability of C. tropicalis in the presence of ISO, the colorimetric assay for the reduction of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium) was performed as described by [54 ]. MTT was dissolved in PBS at 2.5 mg/mL and filtered. The fungal inoculum at a cell concentration of 106 CFU/mL was inoculated into 96-well plates at the previously described ISO concentrations, and the plates were incubated for 24 h at 37 °C. Cells with medium were used as control. Then 50 µL of the MTT solution was added to the cells under evaluation at a concentration of 500 µg/mL. The plates were incubated in the dark for 4 h at 37 °C. The tests were performed in triplicate. Viable cells with metabolic activity convert MTT (yellow color) to formazan, which was solubilized with dimethyl sulfoxide (DMSO) showing a purple color; for this, 50 µL of supernatant is removed from each well and 50 µL of DMSO is added, which was read at a wavelength of 550 nm in a SYNERGY LX microplate reader (Biotek) after shaking in the equipment.
3
SLC16A1 Regulation by miR-22-3p in Bovine Hepatocytes
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Cells were seeded in 24-well plates and cultured until approximately 70% confluent. RNA fragments, including SLC16A1 siRNA, miR-22-3p, and negative control (NC), were transfected into bovine hepatocytes using Lipofectamine 3,000 (Invitrogen, California, United States) following the manufacturer’s instructions, and cultured for 48 h. Luciferase reporter experiments were performed in HEK293T cells. PsiCHECK2-SLC16A1 WT or psiCHECK2-SLC16A1 MUT was co-transfected with NC or a miR-22-3p mimic (GenePharma, Shanghai, China) using Lipofectamine 2000 according to the manufacturer’s instructions. The medium was replaced after 6 h. Transfections were performed using 800 ng luciferase reporter and 80 ng of internal control plasmid pRL-TK (Promega). Relative luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega) and a Synergy LX microplate reader (BioTek, Beijing, China). Bovine hepatocytes were seeded in 6-well plates and cultured until approximately 40% confluent. Bovine hepatocytes were infected by recombinant adenovirus at 100 multiplicity of infection for 48 h.
4
Quantifying 4-1BB Receptor Binding
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ELISA was performed to compare the receptor/ligand and receptor/antibody binding. To get N-glycan-abolished 4-1BB protein, His-tagged 4-1BB protein (Sino Biological, expressed in HEK293 cells) was treated with PNGase F (New England BioLabs) under non-reducing conditions, following the manufacturers’ instruction. The digestion was validated by Coomassie blue staining. Native and deglycosylated 4-1BB were loaded on a Ni-NTA-coated 96-well plate and incubated at room temperature for 30 min. After three washes with PBST (phosphate-buffered saline (PBS)/0.1% Tween-20), human Fc-tagged 4-1BBL (Sino Biological, expressed in HEK293 cells, diluted in PBST) or mouse anti-human 4-1BB antibody (4B4-1, 1:200, BioLegend # 309802, San Diego, CA, USA) was added and incubated for another 30 min, followed by three washes. Plate-bound 4-1BBL or 4B4-1 was incubated with horseradish peroxidase-conjugated anti-human/mouse Fc secondary antibody (Invitrogen, Waltham, MA, USA) and developed with TMB substrate (Cell signaling technology, Danvers, MA, USA). The quantified result was acquired on a Synergy LX microplate reader (BioTek, Winooski, VT, USA) by measuring the OD450 value.
5
Characterization of Novel Monolithic Sorbent
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The photo-initiated copolymerization of SBVI was carried out using UV light at 254 nm and 50 Hz (ALYS Labware, Lausanne, Switzerland). A Zeiss ULTRA 55 field-emission scanning electron microscope (Oberkochen, Germany) was employed for scanning electron microscopy (SEM) and energy-dispersive X-ray spectrometry (EDS) tests of the novel monolith at acceleration voltages of 5 and 15 kV, respectively. A Micromeritics Auto Pore 9500 automatic mercury porosimeter (Norcross, GA, USA) was used to measure pore size distribution. The zeta potential of the resulting monolith was measured using a Malvern Nano-ZS ζ-potential meter (Malvern Panalytical, Malvern, UK). An inverted fluorescence microscope (Olympus, Tokyo, Japan) was used to evaluate the adsorption of FITC-labeled BSA on the monolith. Moreover, the adsorbed protein amount was tested using a Synergy LX microplate reader (BioTek, Winooski, VT, USA). Matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) analysis was performed using a Bruker UltrafleXtreme MALDI TOF/TOF mass spectrometer (Bruker Daltonics, Billerica, MA, USA) to identify the enriched N-glycopeptides. Only peptides containing the Asn-X-Ser/Thr/Cys sequence (X is any amino acids other than Pro) were identified as N-glycopeptides.
6
SARS-CoV-2 Neutralization Assay
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The hMAbs used in this study were generated and purified as described (22 (link)). CB6, REGN10987, and REGN10933 hMAbs were included as controls (25 (link), 26 (link)). To test the neutralizing activity of hMAbs, confluent monolayers of Vero E6 cells (4 × 104 cells/well, 96-plate well format, quadruplicates) were infected (MOI of 0.01 or 0.1) with the indicated rSARS-CoV-2 for 1 h at 37°C. After viral absorption, postinfection media containing 3-fold dilutions of the indicated hMAbs (starting concentration of 500 ng/well) were added to the cells and incubated at 37°C for 48 h. Cells were then fixed in 10% neutral buffered formalin overnight and washed with PBS before fluorescence signal was measured and quantified using a Synergy LX microplate reader and Gen5 data analysis software (Bio-Tek). The mean and SD of viral infections were calculated from individual wells of three independent experiments conducted in quadruplicates with Microsoft Excel software. Nonlinear regression curves and NT50 values were determined using GraphPad Prism Software (San Diego, CA, USA; version 8.2.1). Representative images were captured with an EVOS M5000 imaging system (Thermo Fisher) at ×10 magnification.
7
Quantification of Lung Collagen and Hydroxyproline
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The expression of type I and III collagen major isoforms (Col1a1 and Col3a1) was detected by ELISA kits (E-EL-R0041c and E-EL-R0235c, Elabscience, Wuhan, CN). Five rats for each test group were used. In brief, 100 mg of frozen lung tissue of each rat was ground in liquid nitrogen and resuspended in 1mL PBS. After repeated freeze-thaw, homogenate supernatant was obtained by centrifugation at 5000 g for 5 minutes at 4°C. After that, standard ELISA procedure was carried out according to the manufacturer’s instruction and absorbance at 450 nm was measured by a Synergy LX microplate reader (BioTek, USA).
The hydroxyproline content in lung tissue homogenate was detected by a HYP assay kit (BC0255, Solarbio, Beijing, CN). Five rats for each test group were used. In brief, 200 mg of frozen lung tissue of each rat was cut into very small pieces and digested 4–6 hours at 110°C within 2 mL of digestion buffer. After adjusting pH to 6–8 and making total volume to 4 mL with sterile water, the supernatant was obtained by centrifugation at 16,000 rpm for 20 minutes at 25°C. After that, HYP concentration in the homogenate was determined according to the manufacturer’s instruction and absorbance at 560 nm was measured by a Synergy LX microplate reader (BioTek, USA).
The hydroxyproline content in lung tissue homogenate was detected by a HYP assay kit (BC0255, Solarbio, Beijing, CN). Five rats for each test group were used. In brief, 200 mg of frozen lung tissue of each rat was cut into very small pieces and digested 4–6 hours at 110°C within 2 mL of digestion buffer. After adjusting pH to 6–8 and making total volume to 4 mL with sterile water, the supernatant was obtained by centrifugation at 16,000 rpm for 20 minutes at 25°C. After that, HYP concentration in the homogenate was determined according to the manufacturer’s instruction and absorbance at 560 nm was measured by a Synergy LX microplate reader (BioTek, USA).
8
Quantifying Hydrogen Peroxide in Soybean Roots
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Hydrogen peroxide content was measured using the “Hydrogen Peroxide Assay Kit” (Abcam, Cambridge, England, Ab 102500) based on an OxiRed probe with small modifications. Previously cut roots of soybean seedlings (300–400 mg), frozen in liquid nitrogen and stored at −80 °C, were homogenized in chilled mortars in liquid nitrogen. The homogenized samples were transferred to Eppendorf tubes containing 500 µL of assay buffer and centrifuged (13,000 rcf, 4 °C, for 15 min). Subsequently, 50 µL of the supernatant or provided standard were added to the microplate wells and supplemented with 50 µL of the reaction mixture containing the assay buffer, OxiRed probe and horseradish peroxidase. Due to the rapid color change, absorbance was measured immediately at λ = 570 nm on a Synergy LX microplate reader (BioTek, Wnooski, VT, USA). Each sample was measured in two technical replicates. The analysis was performed in four biological replicates.
9
Dual-Luciferase Assay for Promoter Analysis
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For the dual-luciferase (Dual-LUC) assay, the pGreenII 0800 LUC plasmid (Hellens et al., 2005 (link)) was modified as the effector vector. A Gateway R1R2 cassette and a 66 bp mini 35S promoter were PCR amplified and sequentially inserted into the multicloning site (MCS) upstream of the firefly luciferase gene in the effector vector, to make it compatible for the Gateway cloning technique and capable to test promoter sequences lacking a TATA-box or other essential elements of transcription. The Probe-1MS1 sequence was first TA TOPO (Thermo Fisher Scientific) cloned into pCR8 and LR cloned into the effector vector. The MS188 coding sequence was Gateway cloned into the pUB-DEST vector (Grefen et al., 2010 (link)). All vectors were co-infiltrated into tobacco leaves with p19 of Tomato bushy stunt virus to enhance the transient expression. The infiltrated leaf tissues were collected 3 d after transfection and assayed for the LUC and REN levels using the Promega Dual-Glo® Luciferase Assay Kit (E2920) and BioTek Synergy LX Microplate Reader in accordance with the manufacturer’s instructions.
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