Huh-7 cells at density of 10,000 per well were coated in 96-well cell culture plate overnight to form monolayer adhered cells. According to the pseudo-viral infectivity determine assay mentioned above, viral dilution of relative light unit (RLU) at around 30,000 was used. Three-fold serially diluted antibody or vaccinated plasma was mixed with pseudovirus at ratio of 1:1 for 1 h at 37 °C. The mixture was added and incubated with Huh-7 cells for 12 hours, followed by refresh of the culture medium with fresh DMEM supplied with 10% FBS. The cells were incubated for another 48 hours, washed with PBS for two times, and subsequently lysed with 50 μL of lysis reagent (Promega). 30 μL of cell lysates were added into the substrate of Firefly Luciferase Assay Kit (Promega) and the RLU readout was detected. Percent of inhibition was calculated as relative reduction of RLU compared with the control well (add cells and pseudovirus, without antibody). Data were non-linear fitted and ID50 was calculated by the equation of four parameters regression using GraphPad Prism.
Lysis reagent
Manufactured by Promega
Sourced in United States
Lysis reagent is a laboratory product designed for the disruption and solubilization of cells or tissues. It facilitates the release of cellular components, including proteins, nucleic acids, and other biomolecules, for further analysis or downstream processing.
Automatically generated - may contain errors
Lab products found in correlation
Luciferase assay reagent, by Promega (5 mentions)
Luciferase assay kit, by Promega (4 mentions)
Firefly luciferase assay kit, by Promega (2 mentions)
Modulus luminometer fluorometer, by Promega (2 mentions)
Hexane, by Biosolve (2 mentions)
Dimethyl sulfoxide (dmso), by Biosolve (2 mentions)
Ultra pure ethanol, by Merck Group (2 mentions)
Dulbecco s modification of eagle s medium high glucose dmem without phenol red, by Merck Group (2 mentions)
Glass fiber filters mg 227 1 60, by Sartorius (2 mentions)
Luciferine reagent, by Promega (2 mentions)
17β estradiol, by Merck Group (2 mentions)
Sodium pyruvate, by Merck Group (2 mentions)
Acetonitrile, by Biosolve (2 mentions)
Acetone, by Biosolve (2 mentions)
Counting beads, by Thermo Fisher Scientific (2 mentions)
Ultra 384 luminometer, by Tecan (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Luciferase substrate, by Promega (2 mentions)
29 protocols using lysis reagent
1
Antibody Neutralization Assay with Pseudovirus
2
Dual-Luciferase Reporter Assay for Gene Expression Analysis
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Cells were seeded in 96-well or 12-well plates and transfected using PEI according to the manufacturer’s instructions 48 h after transfection. Cells were lysed with 100 μL of lysis reagent (Promega, Madison, WI, USA). Firefly and Renilla luciferase expression was assessed using a luminometer (BioTek, Winooski, VT, USA) according to the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA). Briefly, after thawing frozen samples at room temperature, 10 μL of lysates was added into a 96-well plate, followed by adding 10 μL of luciferase assay reagent (Promega, Madison, WI, USA). The plate was placed in a luminometer at room temperature. The delay and measurement times were set for 2 and 10 s, respectively. All measurements were finished within 15 min. All the luciferase reporter assays were calculated as the ratio between firefly and Renilla luciferase activities. The pGL3-Rluc/Fluc plasmid contained a Renilla luciferase under the control of T7 promoter. Data generated from three independent experiments were used for comparison.
3
Arabidopsis Protoplast Transformation Assay
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The LUC reporter construct, the effector plasmid, and the 35S:GUS internal control plasmid were co-transformed into Arabidopsis protoplasts. After incubation overnight, the protoplasts were pelleted and proteins were isolated by resuspending the cells in lysis reagent (N1610, Promega). Activities of LUC luminescence and GUS fluorescence were measured using a Modulus luminometer/fluorometer (Promega). The relative activity was quantified as the ratio of LUC/GUS.
4
Murine NSC Proliferation Assay
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Luciferase activity levels from luciferase (luc)+ murine NSCs were used as an indicator of cell proliferation. At 24–72 h post treatment, cell lysates were prepared using the lysis reagent per manufacturer's directions (Promega, Madison, WI). Luciferase assays were performed using 10 µl of cell lysate and 10 µl of the luciferase substrate provided in an opaque 96-well plate. Luminesce levels was measured on a luminometer (Luminoskan, Thermo Fisher Scientific, Walthan MA) using a 10 s read time. NSC growth characteristics that were measured by luciferase activity was repeated in similarly designed, selected experiments using 3[H]-thymidine incorporation assay, as previously described [29] .
5
SARS-CoV-2 Spike Protein Pseudovirus Assay
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Pseudovirus bearing wild or mutant CoVs S protein and a defective HIV-1/VSV genome by which luciferase reporter was produced, as previously described,9 (link) were used to infect target Caco2 cells (HIV-1 backbone-based PsV) and Calu-3 cells (VsV backbone-based PsV) in the presence or absence of each peptide at indicated concentrations, respectively. After 12 h, culture medium was refreshed and cultured an additional 48 h. Then, cells were washed with PBS, lysed with lysis reagent (Promega), and relative light units (RLU) detected by using Luciferase Assay Kits.
6
Estrogen and Androgen Receptor Bioassays
7
Transient Transcription Assay in Arabidopsis
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Five micrograms of the IAA19p:LUC reporter plasmid containing a REN expression cassette was co-transformed with 5 μg of SEU-3HA, SEU4KR-3HA, PIF4-3HA, SEU-3HA and PIF4-3HA, or SEU4KR-3HA and PIF4-3HA effector plasmids into Arabidopsis protoplasts. After overnight incubation in the dark, protoplasts were lysed and resuspended with lysis reagent (Promega). The activities of firefly (Photinus pyralis) and Renilla (Renilla reniformis) luciferases (LUC and REN, respectively) were measured with a Modulus luminometer/fluorometer (Promega). REN was used as a control to monitor the transformation efficiency. Relative LUC levels were expressed as the ratio of LUC/REN.
8
SARS-CoV-2 Pseudovirus Infection Assay
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HIV-1 backbone-based Pseudovirus (PsV) bearing wild or mutant SARS-CoV2 S protein was produced, as previously described [27 (link)]. Caco2 cells were used as target cells and were seeded in wells of a 96-well plate 48 h in advance. PsV in the presence or absence of each peptide at indicated concentrations was added into target cells. After 12 h, culture medium was refreshed and continuously cultured for an additional 48 h. Then, cells were lysed with lysis reagent (Promega) for relative light units (RLU) detection by using Luciferase Assay Kits.
9
Gaussia and Firefly Luciferase Assay
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We used D-PBS (with Ca and Mg) as assay buffer to measure Gaussia luciferase activity in the supernatant of infected cells [26] (link). Coelenterazine was added from a stock solution (in acidified ethanol) to a final concentration of 1.5 µM. To measure Firefly luciferase activity, cells were harvested in Promega Lysis Reagent (25 mM Tris-phosphate pH 7.8, 2 mM DTT, 2 mM 1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid, 10% glycerol, 1% Triton X-100). Measurements were taken in a Hidex Plate Chameleon V instrument with 100 ms delay and 2 s counting time.
10
Muscle Tissue RNA Extraction and cDNA Synthesis
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Approximately 20 mg of muscle tissue was transferred to a 1.5 mL tube (Axygen, Silicon Valley, CA, USA) with 300 μL of lysis reagent (Promega, Madison, WI, USA) and then homogenized three times at 65 Hz for 30 s each. Then, 300 μL of RNA dilution liquid was added to the homogenate and incubated for 5 min until centrifugation at room temperature at 13,000 rpm, and total RNA was extracted, in accordance with the protocol of the manufacturer (Promega). The RNA concentration was detected by a NanoDrop spectrophotometer (Thermo, Waltham, MA, USA), and the quality was assessed by agarose gel electrophoresis. Total RNA was qualified and used for the next manipulation (Figure S1 ). cDNA was synthesized using the PrimeScript RT Reagent Kit with gDNA Eraser (Takara, Shiga, Japan) in a 10 μL volume, in accordance with the instructions of the manufacturer.
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