TZM-bl assays were adapted from Wei et al., 2003 and Montefiori, 2005 [78 (link), 79 (link)]. HIV-1 pseudovirus stocks were generated by transfecting HEK-293T cells. For neutralization assays bNAbs were diluted to 20 µg/mL and a three-fold serial dilution in triplicates was performed in flat-bottom 96 well plates. Pseudovirus at a dilution translating into 20 × RLU of the background control were added to each well, except the cells-only control. After 1 h incubation, 104 TZM-bl cells, containing DEAE dextran, were added to each well and plates were incubated (37 °C, 5% CO2). After 48 h the supernatant was removed, and cells were washed with PBS prior to adding lysis buffer (Promega, cat. #A8261). The plate was kept at − 80 °C overnight to ensure complete virus inactivation. After thawing, the cell lysate was mixed 1:1 with Bright-Glo luciferase substrate (Promega Luciferase Assay System, cat. #E2610) in a black flat bottom 96 well plate. Luminescence was measured using a GloMax plate reader (96 Microplate Luminometer, Promega, USA). IC50s were compared to published data available in the CATNAP database [34 (link)].
96 microplate luminometer
Manufactured by Promega
Sourced in United States
The 96 Microplate Luminometer is a laboratory instrument designed to measure luminescence from 96-well microplates. It is used to quantify light-emitting chemical reactions, such as those involved in bioluminescence and chemiluminescence assays. The luminometer provides sensitive detection and rapid analysis of multiple samples simultaneously.
Automatically generated - may contain errors
Lab products found in correlation
Luciferase assay system, by Promega (2 mentions)
Hep3b, by American Type Culture Collection (2 mentions)
Lysis buffer, by Promega (1 mentions)
Glomax, by Promega (1 mentions)
Pgl3 control vector, by Promega (1 mentions)
Cmv firefly luciferase ires puro lentivirus, by Thermo Fisher Scientific (1 mentions)
Protein assay dye reagent, by Bio-Rad (1 mentions)
96 well plate, by Corning (1 mentions)
Dual luciferase reporter gene assay kit, by Promega (1 mentions)
Neon device, by Thermo Fisher Scientific (1 mentions)
Snu449, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Snu 449, by American Type Culture Collection (1 mentions)
7 protocols using 96 microplate luminometer
1
HIV-1 Pseudovirus Neutralization Assay
2
Luciferase Assay in HeLa Cells
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For Luciferase assays, HeLa cells were transfected with the pGL3-control vector (Promega, Madison, USA), which encodes firefly luciferase gene. pGL3-transfected cells were lysed and processed using the Luciferase Assay System (Promega, Madison, USA) according to the manufacturer's instructions. The light emission was measured in a 96 microplate luminometer (Promega, Madison, USA).
3
Establishing Stable Luciferase-Expressing Cell Lines
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Both Hep3B and SNU-449 cells were first transfected CMV-Firefly luciferase-IRES-Puro lentivirus (Cellomics, USA). Briefly, cells were cultured in a 6-well plate at the concentration of 1×105 cells with 2 mL medium containing 10% heat-inactivated FBS in each well. After 12-hr incubation, polybrene was gently mixed with cells at the concentration of 6 μg/mL for 8 hrs prior to the addition of five multiplicity of infection lentivirus in the fresh medium (EMEM or PRMI-1640) which contains 10% heat-inactivated FBS. Cell selection for both the cell lines was conducted for at least 12 days with puromycin. Stable fluorescence signal was analyzed using a 96 Microplate Luminometer (Promega, USA), and the cells were both characterized by identity verification with short tandem repeat analysis for human cell lines.
4
Mitotane-Induced Caspase-3 Activity in H295R Cells
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After 48 h treatment with mitotane, H295R cells were collected by centrifugation, washed with cold 0.1 M phosphate buffered saline (PBS), and suspended in 600 μL ice-cold lysis buffer [25 mM 4-2-hydroxyethyl-1-piperazineethansulphonic acid (HEPES), 5 mM MgCl2, 5 mM EDTA, 5 mM dithiothreitol, 2 mM phenylmethylsulphonyl fluoride] for 15 min; the suspension was frozen/thawed 10 times (liquid nitrogen/37 °C) and centrifuged at high speed for 30 min at 4 °C and the supernatant was collected. The samples were diluted to achieve a protein concentration of 0.5 mg/mL and incubated for 45 min at 37 °C with 65 μM (final concentration) fluorogenic substrate N-Acetyl-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin (Ac-DEVD-AMC). Caspase-3 activity was evaluated as the release of fluorescent 7-amino-4-methylcoumarin (AMC), using a 96 Microplate Luminometer (GloMax, Promega, Milan, Italy) and recording Δ min fluorescence at 380 nm excitation and 510 nm emission. Fluorescence was referred to AMC standard curve (ranging from 10 nM to 10 µM). Proteins were evaluated with the Bio-Rad protein assay dye reagent (Bio-Rad, Milano, Italy).
5
Transfection Efficiency Evaluation of PLL/DNA/Peptide Polyplexes
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HeLa cells, HEK293 cells, U251 cells and COS7 cells were separately seeded in 96‐well plates (Corning Inc., Lowell, MA, USA) at a density of 5 × 103 cells/well on the day before transfection. When confluence reached 70–80%, the cells were transfected with PLL/DNA or PLL/DNA/peptide polyplexes containing 200 ng of DNA and incubated for 4 hours. The medium was then changed with fresh DMEM growth medium, and the cells were incubated for the reporter gene assays. Transfection mediated by Lipofectamine 2000 was performed in accordance with the manufacturer's instructions. Luciferase gene expression was measured at 24 hours after transfection. Luciferase assays were performed with a Luciferase assay system, and relative light units (RLU) were measured with a 96 Microplate Luminometer (Glomax; Promega). Protein concentrations in the cell extracts were measured by a BCA assay. The final values were calculated as RLU/mg protein and reported as the mean ± SD obtained from triplicate transfections. HEK293 cells was used to evaluate the transfection of PLL/DNA/peptide polyplexes with pCMV‐N‐EGFP plasmid. Images were taken using the DMI 4000B system (Leica Microsystems, Wetzlar, Germany) under a 10× objective.
6
STAT3 Luciferase Reporter Assay
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The luciferase reporter gene pGL-3-STAT3-luciferase (GLSTAT3-Lu) was synthesized by Beijing Yuan Ping Hao Biotechnology Co., Ltd. pGL-3-Basic was used in the control group. After GLSTAT3-Lu/pGL-3-Basic (10 µg/well) and FLAG-CTLA-4/FLAG (10 µg/well) were transfected into CD4+ T cells (2×107 cells) by electroporation methods using a Neon™ device according to the manufacturer's protocol (Invitrogen; Thermo Fisher Scientific, Inc.) for 48 h, luciferase reporter gene activity was assessed using a dual-luciferase reporter gene assay kit, according to the manufacturer's protocol (Promega Corporation). The luciferase detection device used was a 96-microplate luminometer (Promega Corporation) and secreted alkaline phosphatase (cat. no. KA1362; Abnova) was used to normalize luciferase activity. Each experiment was repeated three times. STAT3 and phosphorylated (p)STAT3 protein levels were also monitored via western blotting. Anti-STAT3 (1:5,000; cat. no. ab119352) and anti-pSTAT3 (1:10,000; cat. no. ab76315) were both obtained from Abcam.
7
Establishment of Luciferase-Labeled Liver Cancer Cell Lines
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SNU-449 and Hep3B cell lines were all purchased from American Type Culture Collection (ATCC, USA) in May 2019. Hep3B [5] was cultured in EMEM (ATCC, USA) with 10% fetal bovine serum (FBS, Gibco, USA) and SNU-449 was cultured in RPMI-1640 medium, with 10% heat-inactivated FBS, respectively. Both of them were cultured in the same condition of humidified incubator at 37°C with 5% CO2. Hep3B-luc and SNU-449-luc were then acquired by CMV-Firefly luciferase-IRES-Puro lentivirus transfection. For details, cell selection was conducted for at least 12 days with 1μg/ mL puromycin. Stable fluorescence signal of both cell lines was confirmed by 96 microplate luminometer (Promega, USA). Two cell lines were both characterized using STR (Short Tandem Repeat) analysis for identity verification of human cell lines in 2019.
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