We used the SARS-CoV-2 Surrogate Virus Neutralization Test Kit (Cat. No. L00847) to detect spike-specific neutralizing antibodies (Tan et al., 2020 (link)). To evaluate the level of peptides induced neutralizing antibodies, the S1–61, S1–66, S1–82, and BSA (100 ng/well) were pre-coated in 96-well plate at 4 °C overnight. After washing, 200 µL serum (1:20 diluted with samples dilution buffer) was added, followed by incubation at 37 °C for 30 min. After three rounds of absorption, the supernatant was collected for the analysis of inhibitory ability. Briefly, we mixed 60 µL of negative control, positive control, or samples (preincubated or not) with the HRP-RBD solution in a volume ratio of 1:1 and incubated at 37 °C for 30 min. Next, 100 µL of each mixture was added to the corresponding wells and incubated at 37 °C for 15 min. The plate was washed with washing solution for four times. Then, 100 µL TMB Solution was added to each well and kept in dark at 25 °C for 15 min, followed by adding 50 µL stop solution to each well. The absorbance was read at 450 nm immediately using Synergy™ LX Multi-Mode Microplate Reader ((Bio Tek, VT, USA) with Gen5 software (Version 3.05.11). The inhibitory ability was calculated using the formula: .
Synergy lx multi mode microplate reader
Manufactured by Agilent Technologies
Sourced in United States
The Synergy™ LX Multi-Mode Microplate Reader is a versatile lab equipment that can perform various detection modes for microplate-based assays. It is capable of absorbance, fluorescence, and luminescence measurements.
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Lab products found in correlation
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Cck 8 reagent, by Beyotime (1 mentions)
Udp glca, by Corning (1 mentions)
Udp gal, by Promega (1 mentions)
Udp glo glycosyltransferase assay, by Promega (1 mentions)
Take3 micro volume plate, by Agilent Technologies (1 mentions)
Lysing matrix e tube, by MP Biomedicals (1 mentions)
Inhibitex buffer, by Qiagen (1 mentions)
Qiaamp fast dna stool mini kit, by Qiagen (1 mentions)
Rabbit anti fn polyclonal antibodies, by Merck Group (1 mentions)
Peroxidase conjugated goat anti rabbit immunoglobulin, by Merck Group (1 mentions)
Universal rna purification kit, by EURx (1 mentions)
Take3 plate, by Agilent Technologies (1 mentions)
Rnase free dnase set, by Qiagen (1 mentions)
Inverted microscope, by Leica (1 mentions)
Oil red o, by Merck Group (1 mentions)
Cell counting kit 8 assay, by Dojindo Laboratories (1 mentions)
Celltiter glo luminescent cell viability assay kit, by Promega (1 mentions)
Cisplatin, by Promega (1 mentions)
Celltiter 96 aqueous mts reagent, by Promega (1 mentions)
13 protocols using synergy lx multi mode microplate reader
1
SARS-CoV-2 Spike Protein Neutralization Assay
2
Cell Proliferation Assay with CCK-8
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BGC823 and HGC27 cells were re-suspended with DMEM and placed into 96-well plates (5,000 cells in 200 µl per well). After incubation at 37°C for 0, 24, 48 or 72 h, 10 µl CCK-8 reagent (Beyotime Institute of Biotechnology, Haimen, China) was added to each well. After incubation at 37°C for 2 h, the absorbance at 450 nm was quantitated using a Synergy™ LX Multi-Mode microplate reader (BioTek Instruments, Inc., Winooski, VT, USA).
3
Characterization of GalS1 Glycosyltransferase
4
Plasma EDA-FN Quantification by ELISA
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The level of EDA-FN was determined in plasma samples using a human cellular fibronectin ELISA kit (Human cFN ELISA Kit, Wuhan Fine Biotech Co., Ltd., Wuhan, China). Each sample was analyzed twice and the concentration was calculated from the calibration curve prepared according to the manufacturer’s protocol. The mean value of the two readings was used for statistical analysis. A Synergy LX Multi-Mode Microplate Reader was used to measure EDA-FN (BioTek, Winooski, VT, USA).
5
Glycerol Quantification for Lipolysis Assessment
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The glycerol concentration was quantified on day 9 of the trial using an adipolysis assay kit (MAK313; Sigma-Aldrich, St. Louis, MO, USA) to compare the lipolysis rates among the studied groups. Each sample was measured twice, in an amount of 10 µL, according to the protocol provided by the manufacturer. Optical density (OD) at a 570 nm wavelength with a correction of 630 nm was measured (Synergy™ LX Multi-Mode Microplate Reader, BioTek Instruments, Inc., USA). Glycerol concentration was calculated in µg/mL according to the OD of the standards and the standard curve. Then, the percentage of lipolysis was assessed relative to DC.
6
MTT Assay for Cell Viability
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CT26 cells were seeded and treated with siBmal1 (or siNC as the control) for 48 h. The culture medium was replaced with fresh medium containing MTX (at a series of concentrations, n = 6), and allowed for incubation for another 2 days. Then, the cells were incubated with MTT (1 mg/ml, dissolved in serum-free medium) for 4 h. Cell residues were dissolved with 200 μl DMSO. The absorbance at 490 nm was determined using Synergy LX Multi-Mode Microplate Reader (BioTek, Winooski, VT, USA).
7
Microbial DNA Extraction from Rumen, Fecal, and Milk Samples
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Microbial DNA from the ruminal, fecal, and milk samples was extracted following the procedures previously described by Welch et al. (2020) (link) with modifications. Briefly, 350 μl of rumen sample, 0.35 g of fecal sample, or 1,000 μl of milk sample were placed in 2-ml Lysing Matrix E tubes (MP Biomedicals LLC, Irvine, CA, United States), which are homogenized using a QIAGEN vortex adapter (QIAGEN, Venlo, Netherlands) to disrupt the cells. Enzymatic inhibition was achieved by using InhibitEX Buffer (QIAGEN, Venlo, Netherlands), and DNA elution and purification were carried out using a spin column and a series of specialized buffers according to manufacturer’s specifications (QIAamp Fast DNA Stool Mini Kit; QIAGEN, Venlo, Netherlands). Calculation of DNA concentration and purity in the resulting eluate was performed spectrophotometrically using the Synergy LX Multi-Mode Microplate Reader in conjunction with the Take3 Micro-Volume Plate (BioTek Instruments Inc; Winooski, VT, United States). Samples with a minimum volume of 90 μl and 10 ng/μl of DNA were stored at −80°C until the following day. Samples that failed to meet these requirements were rejected and subjected to a new DNA extraction cycle.
8
Quantification of Plasma Fibronectin by ELISA
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The level of pFN was measured by an enzyme-linked immunosorbent assay (ELISA), using a well-defined domain-specific monoclonal antibody directed to the central cell-binding domain of FN (FN30-8; M010 TaKaRa Shuzo Co. Ltd., Shiga, Japan), as previously described [18 (link)]. The monoclonal anti-FN antibodies were used as a coating agent. The amount of FN bound by the monoclonal antibody was quantified using rabbit anti-FNpolyclonal antibodies (Sigma Chemical Co., St. Louis, MO, USA) and peroxidase-conjugated goat anti-rabbit immunoglobulins (Sigma Chemical Co., St. Louis, MO, USA) as the secondary antibodies. Each sample was analyzed twice and the concentration of pFN was calculated from the calibration curve; the mean value of the two readings was used for the statistical analysis. The pFN concentration was detected with a Synergy LX Multi-Mode Microplate Reader (BioTek, Winooski, VT, USA). A human pFN preparation (Sigma, St. Louis, MO, USA) was used as a standard for determining the FN-ELISA.
9
RNA Extraction, Purification, and RT-qPCR Analysis
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The total cellular RNA was extracted using a Universal RNA Purification Kit (EURx, Gdańsk, Poland) per the manufacturer’s protocol. BioTek’s Take3 plate measured RNA purity and quantity, Synergy™ LX Multi-Mode Microplate Reader (BioTek, Santa Clara, CA, USA) at 260 and 280 nm absorbance. All RNA samples were purified from genomic DNA using RNAse-Free DNase Set (Qiagen, Germany) and were reverse-transcribed using a RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, Waltham, MA, USA), following the manufacturer’s instructions. The analysis of the expression levels of the housekeeping genes, 18S, HPRT, and GAPDH, was performed with real-time qPCR thermocycler Gentier 96E (Xi’an Tialong Science and Technology, Xi’an, China) using KAPA SYBR FAST Master Mix (2X) (Roche) in duplicates with two-step qPCR. The amplification of cDNA was performed following the manufacturer’s conditions.
10
Quantifying Adipocyte Lipid Accumulation
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To investigate the phenotype of mature adipocytes from the various experimental groups, images were obtained after Oil Red O (Sigma-Aldrich, St. Louis, MO, USA) staining to visualize the intracellular lipid droplets. Subsequently, we extracted the dye with isopropanol and quantified the neutral lipid accumulation in adipocytes using the method described by Yang et al. [62 (link)]. The supernatants from the 12-well plates were removed, and the cells were washed 3 times with phosphate-buffered saline (PBS). Next, the adipocytes were fixed in 10% (v/v) neutral buffered formalin for 10 min, washed with isopropanol for 5 min, and stained for 30 min with a working solution of Oil Red O, prepared ex tempore. The stained wells were then kept in dd H2O and observed using a Leica Inverted Microscope with a 5-megapixel-resolution DMi1 camera. The accumulated Oil Red O dye in the adipocytes was extracted with 100% isopropanol for 10 min. The optical density of the extracts was assessed spectrophotometrically at a wavelength of 490 nm using a Synergy™ LX Multi-Mode Microplate Reader (BioTek Instruments, Inc., USA). Finally, the data were expressed relative to the DC as a percentage.
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