Pseudovirus particles were generated by co-transfection of individual plasmids encoding HIV-1 Tat, HIV-1 Gag/Pol, HIV-1 Rev, luciferase followed by an IRES and ZsGreen, and the SARS-CoV-2 spike protein as previously described(Crawford et al., 2020 (link)). In brief, HEK293T cells were transfected with the pseudovirus encoding plasmids using FuGENE 6 Transfection Reagent (Promega). The virus culture supernatant was harvested at 48 h and 72 h post transfection and stored at −80°C until use. Each virus batch was titrated by infecting 293T-ACE2 and after a 48 h incubation period at 37°C and 5% CO2, luciferase activity was determined after addition of luciferin/lysis buffer (10 mM MgCl2, 0.3 mM ATP, 0.5 mM Coenzyme A, 17 mM IGEPAL (all Sigma-Aldrich), and 1 mM D-Luciferin (GoldBio) in Tris-HCL) using a microplate reader (Berthold). An RLU of approximately 1000-fold in infected cells versus non-infected cells was used for neutralization assays.
Microplate reader
Manufactured by Berthold Technologies
Sourced in Germany, United States
The Microplate reader is a versatile laboratory instrument that measures the absorbance or fluorescence of samples in a microplate format. It is designed to provide accurate and precise quantitative analysis of various biological and chemical assays.
Automatically generated - may contain errors
Lab products found in correlation
D luciferin, by GoldBio (3 mentions)
Fugene 6 transfection reagent, by Promega (3 mentions)
Igepal, by Merck Group (3 mentions)
Coenzyme a, by Merck Group (3 mentions)
Passive lysis buffer (plb), by Promega (2 mentions)
Cell proliferation elisa brdu kit, by Roche (1 mentions)
Vybrant cell adhesion assay kit, by Thermo Fisher Scientific (1 mentions)
Raw264.7 cell, by American Type Culture Collection (1 mentions)
Dcfh da, by Merck Group (1 mentions)
Mtt assay kit, by Abcam (1 mentions)
Psicheck 2 vector, by Promega (1 mentions)
X tremegene 9 dna transfection reagent, by Roche (1 mentions)
Colorimetric ache assay kit, by Abcam (1 mentions)
Phenol red, by Thermo Fisher Scientific (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Beyotime (1 mentions)
Cck 8 reagent, by Dojindo Laboratories (1 mentions)
Donkey serum, by Jackson ImmunoResearch (1 mentions)
Donkey serum, by Santa Cruz Biotechnology (1 mentions)
40 protocols using microplate reader
1
SARS-CoV-2 Spike Protein Pseudovirus Production
2
Cell Proliferation Assay with BrdU
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A total of 4,000 cells were plated in a 96-well plate. After 24 h, the cells were incubated in low-serum media and treated with drugs for 2 days. On the third day, BrdU was added at a final concentration of 1 μM. After incubation for 16 h, DNA synthesis was assayed using a Cell Proliferation ELISA BrdU kit (Roche Molecular, Biochemical, Meylan, France). Newly synthesized BrdU-DNA was determined using a microplate reader (Berthold Technologies, Thoiry, Yvelines, France).
3
SARS-CoV-2 Pseudovirus Neutralization Assay
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Lentivirus-based pseudovirus expressing the SARS-CoV-2 Wu01 spike protein (EPI_ISL_40671) was produced in 293-T cells FuGENE-6 transfection reagent (Promega) and supernatants were harvested and stored at −80 °C. For testing SARS-CoV-2 neutralizing activity, serial dilutions of serum (heat inactivated at 56 °C for 45 min) were co-incubated with pseudovirus supernatants for 1 h at 37 °C and thereafter, 293T cells engineered to express ACE2 were added [25 (link)]. After 48 h of incubation at 37 °C and 5% CO2, luciferase activity was determined after addition of luciferin/lysis buffer (10 mM MgCl2, 0.3 mM ATP), 0.5 mM Coenzyme A, 17 mM IGEPAL (all Sigma-Aldrich, St. Louis, MO, USA), and 1 mM D-Luciferin (GoldBio, St. Louis, MO, USA) in Tris-HCL) using a microplate reader (Berthold). After subtracting background relative luminescence units (RLUs) of un-infected cells, the 50% Inhibitory dose (ID50) was determined as the serum dilution with 50% RLU reduction compared to untreated virus control wells. Every serum sample was measured on different days in two independent experiments, and the mean ID50 values are presented.
4
Pseudovirus Production and Titration
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Pseudovirus particles were generated by co-transfection of individual plasmids encoding HIV-1 Tat, HIV-1 Gag/Pol, HIV-1 Rev, luciferase followed by an IRES and ZsGreen, and the SARS-CoV-2, (Crawford et al., 2020 (link)), SARS-CoV-1 or WiV-1 spike protein. In brief, HEK293T cells were transfected with the pseudovirus encoding plasmids using FuGENE® 6 Transfection Reagent (Promega). The virus culture supernatant was harvested at 48 h and 72 h post transfection and stored at −80°C until use. Each virus batch was titrated by infecting 293T-ACE2 and after a 48 h incubation period at 37°C and 5% CO2, luciferase activity was determined after addition of luciferin/lysis buffer (10 mM MgCl2, 0.3 mM ATP, 0.5 mM Coenzyme A, 17 mM IGEPAL (all Sigma-Aldrich), and 1 mM D-Luciferin (GoldBio) in Tris-HCL) using a microplate reader (Berthold). Pseudovirus dilution resulting in a RLU of approximately 1000-fold in infected cells versus non-infected cells was used for neutralization assays.
5
MTT Assay for Cell Viability
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Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. A549 cells were cultured in a 96-well plate with the selected compound (0–10 μM) or extract (0–100 μg/mL). After a 3-day incubation, 30 μL of 0.5% MTT solution was added to each well. After incubation for 3 h, the supernatant was replaced with 100 μL of methanol with 1% Triton X-100, and then vortexed to dissolve the formazan. Absorbance at 565 nm and reference at 655 nm were measured using a microplate reader (Berthold Technologies, Bad Wildbad, Germany). The half-maximal cytotoxicity concentration (CC50) was calculated using the Reed and Muench method (Reed and Muench, 1938 ).
6
Quantifying Cell Adhesion via Fluorescence
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Cell adhesion was measured using the Vybrant™ Cell Adhesion Assay Kit according to the manufacturer’s instructions (Thermo Fisher Scientific, Rockford, IL, USA). The cells, treated with ALA (0–10 mM) for 24 h, were loaded with calcein acetoxymethyl ester AM (5 µM) at 37 °C for 30 min. Calcein AM is non fluorescent, but, once loaded into cells, is cleaved by endogenous esterases to produce highly fluorescent calcein. Then, 100 μL of the calcein-labeled cell suspension was added to prepared microplate wells with ECM proteins, which was then incubated at 37 °C for 120 min. Non-adherent calcein-labeled cells were removed by careful washing, and the fluorescence was measured using a microplate reader (absorbance maximum of 494 nm and emission maximum of 517 nm, Berthold Technologies GmbH & Co. KG, Bad Wildbad, Germany).
7
Evaluating YYH's Anti-Inflammatory Effects
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RAW 264.7 cells were obtained from American Type Culture Collection (Manassas, VA, USA) and cultured in DMEM which was supplemented with 10% FBS, 1% penicillin-streptomycin solution and 1% GlutaMAX™-1 at 37 °C in a humidified incubator containing 5% CO2. Cells were seeded in 96-well plates at a density of 1 × 105 cells/mL and incubated for 24 h. They treated with different concentrations of YYHs (12.5, 25, 50, 75 μg/mL) in the absence or presence of 1 μg/mL LPS for 24 h, respectively. Then, 50 μL MTT solution (0.5 mg/mL in DPBS) was added to plates and incubated for 4 h in the incubator. After incubation finished, the supernatants was discarded and replaced with DMSO (200 μL) to dissolve the formazan crystal. The absorbance of each well was detected at 570 nm using a microplate reader (BERTHOLD Technologies, Germany).
8
Quantifying Collagen Content in Tissues
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Collagen content was detected by picrosirius red staining followed by counter staining using haemotoxylin. Sirius red staining was quantified by dissolving the stain using 0.5 N Sodium Hydroxide (0.5 N NaOH) and the absorbance was measured at 560 nm using a Microplate Reader (Berthold technologies, USA). Protein content was determined by the Bicinchoninic Acid (BCA) assay using Pierce BCA Protein Assay Kit (Pierce, USA) with different concentration of bovine serum albumin (BSA) (Pierce, USA) as standards. The absorbance was read at 562 nm after incubation at 37°C for 20–30 min using Microplate Reader.
9
Antioxidant Activity Assay Using DPPH
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An antioxidant activity assay was carried out using the DPPH method (1,1-diphenyl-2- picryl hydrazyl). [1 (link), 17 , 18 ] Various extracts with a final concentration of 1000 μg/mL were diluted using the two-fold dilution method (1000–31.25 μg/mL) in a 96-well clear polystyrene microplate. Fifty μL of the sample was mixed with 80 μL of 80 μg/mL DPPH, and the mixture was incubated in the dark for 30 minutes. Absorbance was measured at 520 nm using a Berthold microplate reader. As a positive control, ascorbic acid was subjected to the same method. The assay was carried out in triplicate, and the data were reported as mean ± standard deviation.
The % Inhibition value is calculated by the following formula:
IC50 analysis was performed using GraphPad Prism 9 software, where A0 is the absorbance of the DPPH radical solution without the sample and As is the absorbance of the sample with DPPH radical solution. The assay was carried out in triplicate, and the data were reported as mean ± standard deviation.
The antioxidant activity index (AAI) is calculated as follows:
The % Inhibition value is calculated by the following formula:
IC50 analysis was performed using GraphPad Prism 9 software, where A0 is the absorbance of the DPPH radical solution without the sample and As is the absorbance of the sample with DPPH radical solution. The assay was carried out in triplicate, and the data were reported as mean ± standard deviation.
The antioxidant activity index (AAI) is calculated as follows:
10
Quantifying Cellular Oxidative Stress
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Cellular ROS were evaluated using the fluorescent probe 2′,7′-Dichlorofluorescein diacetate (DCFH-DA Sigma Aldrich). DCHF-DA is an apolar molecule that diffuses easily in cells, where by two successive enzymatic reactions, it is transformed into DCF, a highly fluorescent molecule that is emitted at a wavelength of 532 nm. The oxidation of DCHF to DCF occurs mainly by H2O2. Therefore, the fluorescence intensity is considered directly proportional to the quantity of H2O2 produced by the cells. In our experiments, ECs were treated with 200 µL of 10 μM DCFH-DA for 30 min at 37 °C in the darkness. Production of ROS was measured by a microplate reader (Berthold, Bad Wildbad, Germany) at an excitation wavelength of 485 nm and an emission wavelength of 530 nm.
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