MFB‐F11 cells were cultured as described previously (Tesseur et al, 2006 (link)). In brief, 20 000 MFB‐F11 cells were seeded per well in 96‐well plate until 70% confluency. Cells were washed twice with 1× PBS, followed by incubation with medium from the co‐culture experiments for 24 h at 37°C, 5% CO2. SEAP assay was carried out using Great EscAPe SEAP Chemiluminescence kit (Takara, 631738) according to the manufacturer’s instruction.
Great escape seap chemiluminescence kit
Manufactured by Takara Bio
Sourced in United States
The Great EscAPe SEAP Chemiluminescence Kit is a laboratory product designed to detect and quantify the expression of secreted alkaline phosphatase (SEAP) in cell culture samples. The kit utilizes a chemiluminescence-based detection method to measure SEAP activity, providing a sensitive and reliable way to monitor gene expression in various experimental settings.
Automatically generated - may contain errors
Lab products found in correlation
Hnf4α, by OriGene (2 mentions)
Pcmv lacz plasmid, by Takara Bio (2 mentions)
Victor3, by Takara Bio (2 mentions)
Glomax 20 20 luminometer, by Promega (1 mentions)
Naphthol as mx phosphate, by Merck Group (1 mentions)
Fast blue bb salt, by Merck Group (1 mentions)
E6080, by Promega (1 mentions)
Ascorbic acid, by Solarbio (1 mentions)
β glycerophosphate, by Solarbio (1 mentions)
Benzonase, by Merck Group (1 mentions)
13 protocols using great escape seap chemiluminescence kit
1
Assessing MFB-F11 Cell Response
2
ALP Activity Quantification Protocol
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ALP activity was analyzed by a modified Great EscAPe SEAP chemiluminescence kit (Takara). Each assay condition was performed in triplicate, and the results were repeated in three independent experiments. ALP activity was normalized by the total cellular protein concentrations among the samples.
3
Investigating CTGF Promoter Activity Regulation
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A reporter plasmid carrying secreted alkaline phosphatase (SEAP) under the control of human CTGF promoter was a kind gift from Dr. Andrew Leask (Western University, Canada). Plasmids carrying Myc‐DDK tagged cDNAs for murine Yap (GeneBank accession# NM_001171147), and Hnf4α (NM_008261) were purchased (OriGene, Rockville, MD). These plasmids, empty pCMV6 vector or in combination (100 ng per well) were transfected into HEK293 or HepG2 cells in 24‐well plates. To knockdown human Hnf4α, 50 nM Stealth RNA oligonucleotides or non‐targeting scramble control was also transfected. One day after transfection, media were switched to conditioned media containing with or without Tgf‐β1 (5 ng/mL). A pCMV‐lacZ plasmid (Clontech) at 20 ng/well was transfected as internal control for normalization based on β‐galactosidase activities in the co‐transfected cells according to Leask et al.27 The SEAP activity was measured in conditioned medium 48 hours later using the Great EscAPe SEAP Chemiluminescence kit (Clontech, Mountain View, CA) according to the manufacturer’s instructions. SEAP activities were measured in triplicate experiments and relative CTGF promoter activity was expressed as fold change in comparison to the normalized SEAP activities of vector controls.
4
Screening Anti-HPV16 Extract Activity
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The 293TT cells were grown in 96-well plates at 5 × 103 cells/well for 24 h. Different extract concentrations were added to the cells for 16 h. The cells were gently washed with PBS once to remove the remaining extract inside the wells. The cells were infected with 1 × 105 relative light units (RLU) of HPV16-SEAP PVs for 1 h. The medium was removed and replaced by a complete culture medium. The cultures were incubated for 48 h at 37°C under 5% CO2, and then the SEAP content in 10 μl of clarified cell supernatant was determined using the Great EscAPe SEAP Chemiluminescence Kit (Clontech, USA). Each concentration of the extract was assayed for virus inhibition in triplicate.
5
HPV Pseudovirion Neutralization Assay
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HEK 293 cells (1.5 × 105 cells/well) were pre-plated in a 96-well plate with 100 µL DMEM containing 1% penicillin-streptomycin and 10% FBS for 4–6 h. Purified HPV 16 or HPV 18 pseudovirions were subjected to a 2000-fold dilution. The diluted pseudovirion stock (60 µL/well) was added with 60 µL series diluted serum (1:20 to 1:327,680) in a 96-well non-treated plate and then incubated at 4 °C for 1 h. Subsequently, 100 µL of the pseudovirion-serum mixture was transferred onto the pre-plated cells, followed by incubation for 72 h at 37 °C under a 5% CO2 atmosphere. After that, 100 µL of supernatant was collected and centrifuged at 500× g for 5 min. The SEAP content in 15 µL of the supernatant was measured using the Great ESCAPE SEAP Chemiluminescence Kit (Clontech, CA, USA). After adding the substrate for 20 min, each sample was analyzed using a 1420 multilabel counter (VICTOR3). Neutralization activity was determined by identifying the highest serum dilution that led to a >50% reduction in SEAP activity.
6
Characterizing Lentiviral Pseudoviruses for Vaccine Testing
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In order to use the BPV PsVs to perform vaccine testing in PBNAs, it was first necessary to demonstrate that the PsVs were able to infect mammalian cells and to transfer and express their encapsidated reporter gene to the cells. To this end, HEK293TT cells were trypsinised, resuspended in complete DMEM (cDMEM) medium, and counted using a haemocytometer. Cells were seeded in a Corning® Costar® 24-well plate (Merck) at 1.5 × 105 cells/well, and incubated overnight at 37 °C, 5% CO2. PsV fractions were diluted 1:10 in cDMEM and added to the cells in triplicate at 500 μL/well. The cells were further incubated for 72 h, after which SEAP activity was assayed using the Great EscAPe SEAP Chemiluminescence Kit (Clontech Laboratories, Inc., Mountain View, CA, USA) as per the manufacturer’s instructions, with dilutions and reagents modified to 0.6× the volumes specified (60 uL total reaction volume). SEAP signal was read using a GloMax® 20/20 Luminometer (Promega).
7
Quantitative Assessment of Alkaline Phosphatase and Mineralized Matrix Nodules
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ALP activity was assessed using a modified Great EscAPe SEAP Chemiluminescence kit (Clontech Laboratories, Inc., Mountain View, CA, USA) and/or histochemical staining assay [using a mixture of 0.1 mg/ml naphthol AS-MX phosphate (Sigma-Aldrich, St. Louis, MO, USA) and 0.6 mg/ml Fast Blue BB salt (Sigma-Aldrich)] as described (18 (link)). For quantitative ALP measurement, the ALP activity was determined at a wavelength of 405 nm (E6080; Promega Corporation) in triplicate and the results were repeated in at least three independent experiments.
ARS staining was conducted to evaluate mineralized matrix nodules as described previously (19 (link)). Briefly, cells were cultured in the presence of ascorbic acid (50 µg/ml; Beijing Solarbio Science and Technology Co., Ltd., Beijing, China) and β-glycerophosphate (10 mmol/l; Beijing Solarbio Science and Technology Co., Ltd.). Cells were fixed with 0.05% (v/v) glutaraldehyde (Chongqing Chuandong Chemical Group Co., Ltd., Chongqing, China) at room temperature for 10 min. Subsequent to washing with distilled water, the fixed cells were incubated with 0.4% ARS (Sigma-Aldrich) for 5 min, followed by extensive washing with distilled water. The staining of calcium mineral deposits was recorded under bright field microscopy (T-DH; Nikon Corporation, Tokyo, Japan).
ARS staining was conducted to evaluate mineralized matrix nodules as described previously (19 (link)). Briefly, cells were cultured in the presence of ascorbic acid (50 µg/ml; Beijing Solarbio Science and Technology Co., Ltd., Beijing, China) and β-glycerophosphate (10 mmol/l; Beijing Solarbio Science and Technology Co., Ltd.). Cells were fixed with 0.05% (v/v) glutaraldehyde (Chongqing Chuandong Chemical Group Co., Ltd., Chongqing, China) at room temperature for 10 min. Subsequent to washing with distilled water, the fixed cells were incubated with 0.4% ARS (Sigma-Aldrich) for 5 min, followed by extensive washing with distilled water. The staining of calcium mineral deposits was recorded under bright field microscopy (T-DH; Nikon Corporation, Tokyo, Japan).
8
HPV Pseudovirus Neutralization Assay
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Neutralizations assays were performed using SEAP-expressing pseudoviruses (PV16, PV18, and PV58) according to a previously described method [24] (link). Briefly, OptiPrep-purified, SEAP-expressing HPV 16, 18, and 58 pseudoviruses were diluted 3,000-fold and incubated on ice for 1 hour with 3-fold serial dilutions of mouse sera. 293TT cells were infected by incubating with each pseudovirus–antibody mixture for 3 days. The SEAP content in 10 ul of clarified cell supernatant was determined using a Great EscAPe SEAP Chemiluminescence Kit (Clontech, Mountain View, CA, USA). Neutralization titers were defined as the reciprocal of the highest serum dilution that caused at least a 50% reduction in SEAP activity.
9
CTGF Promoter Activity Regulation by Yap and Hnf4α
10
Neutralization Assay for HPV Pseudoviruses
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Neutralization assays were performed using SEAP-expressing HPV16 PVs according to a previously described method [14 (link)]. Briefly, OptiPrep-purified SEAP HPV16, -18, and -58 PVs were diluted 3,000-fold and incubated on ice for 1 hour with 3-fold serial dilutions of serum. 293TT cells were infected by incubating with each PV–antibody mixture for 72 hours. The SEAP content in 10 μl of clarified cell supernatant was determined using the Great EscAPe SEAP Chemiluminescence Kit (Clontech, Mountain View, CA, USA). Neutralization titers were defined as the reciprocal of the highest serum dilution that caused at least a 50% reduction in SEAP activity.
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