Sf9 cells were seeded in 12-well plates and infected with recombinant baculoviruses expressing the luciferase. Cells infected with a baculovirus not expressing the luciferase were used for the baseline correction. 5 μM MG-132 was added to block the proteolytic activity of proteasome complex 9. The same volume of DMSO was added in parallel as the untreated control. The cells were harvested and lysed with cell lysis reagent (Promega) at 36 hpi. ELISA plates were coated with 30 μg/mL cell lysate diluted in 100 μL of 50 mM sodium carbonate buffer (pH 9.6) at 4°C overnight. The plates were washed three times with PBS containing 0.05% Tween-20 (PBST) and blocked with 5% nonfat milk in TBST buffer for 1 hour at 37°C. Anti-Luciferase polyclonal antibody (Promega) at the dilution of 1:250 was added and incubated at 37°C for 1 hour. After washing three times, 100 μL HRP-conjugated rabbit anti-goat IgG (diluted 1:2,000 in blocking buffer) was added to each well and incubated at 37°C for 1 hour. After washing with TBST, 50 μL of TMB was added and incubated in the dark at 37°C for 25 min. The reaction was stopped by adding 50 μL of 2M H2SO4, and the absorbance was read at 450 nm. All measurement was repeated three times.
Cell lysis reagent
Manufactured by Promega
Sourced in United States
The Cell Lysis Reagent is a solution designed to facilitate the disruption and solubilization of cells. It is a key component for the extraction of cellular contents, including proteins, nucleic acids, and other biomolecules, for further analysis and downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Santa Cruz Biotechnology (2 mentions)
Luciferase assay reagent, by Promega (2 mentions)
Anti luciferase polyclonal antibody, by Promega (1 mentions)
Luciferase assay substrate, by Promega (1 mentions)
Ly294002, by Merck Group (1 mentions)
Pcmv β gal, by Takara Bio (1 mentions)
Luciferase reporter assay kit, by Promega (1 mentions)
Chemiluminescence, by Thermo Fisher Scientific (1 mentions)
P erk, by Santa Cruz Biotechnology (1 mentions)
Complete mini protease inhibitor cocktail, by Roche (1 mentions)
Bradford reagent, by Bio-Rad (1 mentions)
Luminometer, by Berthold Technologies (1 mentions)
Dual luciferase assay kit, by Promega (1 mentions)
Sirius luminometer, by Berthold Technologies (1 mentions)
Elisa kit, by ZeptoMetrix (1 mentions)
Modified great escape seap chemiluminescence assay, by BD (1 mentions)
Steady glo substrate, by Promega (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
X tremegene hp dna transfection reagent, by Roche (1 mentions)
Lipofectamine 2000, by Roche (1 mentions)
14 protocols using cell lysis reagent
1
Quantification of Luciferase Activity in Baculovirus-Infected Sf9 Cells
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Sf9 cells were seeded in 12-well plates and infected with recombinant baculoviruses expressing the luciferase. Cells infected with a baculovirus not expressing the luciferase were used for the baseline correction. 5 μM MG-132 was added to block the proteolytic activity of proteasome complex 9. The same volume of DMSO was added in parallel as the untreated control. The cells were harvested and lysed with cell lysis reagent (Promega) at 36 hpi. ELISA plates were coated with 30 μg/mL cell lysate diluted in 100 μL of 50 mM sodium carbonate buffer (pH 9.6) at 4°C overnight. The plates were washed three times with PBS containing 0.05% Tween-20 (PBST) and blocked with 5% nonfat milk in TBST buffer for 1 hour at 37°C. Anti-Luciferase polyclonal antibody (Promega) at the dilution of 1:250 was added and incubated at 37°C for 1 hour. After washing three times, 100 μL HRP-conjugated rabbit anti-goat IgG (diluted 1:2,000 in blocking buffer) was added to each well and incubated at 37°C for 1 hour. After washing with TBST, 50 μL of TMB was added and incubated in the dark at 37°C for 25 min. The reaction was stopped by adding 50 μL of 2M H2SO4, and the absorbance was read at 450 nm. All measurement was repeated three times.
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Sf9 cells were seeded in 12-well plates and infected with recombinant baculoviruses expressing the luciferase. Cells infected with a baculovirus not expressing the luciferase were used for the baseline correction. 5 μM MG-132 was added to block the proteolytic activity of proteasome complex 9. The same volume of DMSO was added in parallel as the untreated control. The cells were harvested and lysed with cell lysis reagent (Promega) at 36 hpi. ELISA plates were coated with 30 μg/mL cell lysate diluted in 100 μL of 50 mM sodium carbonate buffer (pH 9.6) at 4°C overnight. The plates were washed three times with PBS containing 0.05% Tween-20 (PBST) and blocked with 5% nonfat milk in TBST buffer for 1 hour at 37°C. Anti-Luciferase polyclonal antibody (Promega) at the dilution of 1:250 was added and incubated at 37°C for 1 hour. After washing three times, 100 μL HRP-conjugated rabbit anti-goat IgG (diluted 1:2,000 in blocking buffer) was added to each well and incubated at 37°C for 1 hour. After washing with TBST, 50 μL of TMB was added and incubated in the dark at 37°C for 25 min. The reaction was stopped by adding 50 μL of 2M H2SO4, and the absorbance was read at 450 nm. All measurement was repeated three times.
2
Measuring Virus Entry with pH Modulation
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Cells were plated at 5 × 104 cells in 24-well plates, and 33-(SE)Fluc2 at MOI 1 was adsorbed to cells for 1 hr at 4°C. Unattached virus was removed by rinsing and then cells were treated with buffers at pH 3, 4, 5, 6, and 7 for 3 min at 37°C. After rinsing, cells were incubated in growth media for 1 hr at 37°C and then harvested after adding 150 μL cell lysis reagent (Promega) for 30 min at room temperature. Then, 20 μL of this lysate was mixed with 100 μL luciferase assay substrate (Promega) and read on a spectrophotometer (Tecan) in a 96-well plate. 33-(SE)Fluc2 entry was also measured in the presence of an Akt inhibitor, LY294002 (Sigma-Aldrich, St. Louis, MO) following a similar protocol. Following treatment with buffer either pH 4 or pH 7 for 3 min at 37°C, cells were washed and incubated with growth media containing 0 μM, 25 μM, or 50 μM of LY294002 for 1 hr at 37°C. Luciferase assay was then completed as outlined above.
3
RA Regulation of NELL2 Transcription
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Site-directed mutagenesis was used to mutate each of the two half-RA response elements (RAREs) identified in the mouse NELL2 (mNELL2) promoter (Fig. S1 ). The mutant promoters were generated from wild-type mNELL2 promoter [13] (link) as the template. Mutagenesis was carried out using QuickChange-XLsite-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer's instructions. The primers used for mutagenesis were as follows: a primer set for deleting one half-RARE, 5′-GAA TCC CCT TGC CTT GCC CTT TTG CTG CTG TGT AG-3′ and its complementary sequence (CS), located at the NELL2 promoter (−1047); a set for deleting another half-RARE, 5′-GTC CCC GCA GGT CCC CAG AGC CGG CTG CGG CC-3′ and its CS, located at −223 of the NELL2 promoter. Mutants were verified by DNA sequencing. To determine whether RA regulates NELL2 transcription, the P19 cells were transiently transfected with mNELL2 promoter (NELL2-P)-luciferase reporter constructs (NELL2-pGL3) using Lipofectamine/PLUS reagent. After 24 h, the cells were treated with 1 µM RA for 24 h and lysed with Cell Lysis Reagent (Promega Corp., Madison, WI). Luciferase assays were performed using a luciferase reporter assay kit (Promega Corp.). The transfection efficiency of each assay was normalized by cotransfecting the plasmid p-CMV-β-gal (Clontech, Palo Alto, CA) at 60 ng/ml.
4
Protein Expression Analysis in HeLa Cells
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Total proteins from HeLa cells were extracted with cell lysis reagent (Promega, Madison, WI, USA), and supplemented with Complete Mini protease in hibitorcocktail (Roche Diagnostics, GmbH, Germany). The protein samples were loaded on and separated by SDS-PAGE, after being quantitatively determined by using Bradford Reagent (Bio-Rad, Hercules, CA, USA), and then were transferred to PVDF membranes, which were then blocked in 5% skimmed milk for 1 h at room temperature and probed with an antibody to KLF5, TNFRSF11a, PCNA, Akt, P-Akt, p38, P-p38, Erk, P-Erk or β-actin (Santa Cruz Biotechnology, SantaCruz, CA, USA). Antibody binding was detected by using chemiluminescence (Thermo Scientific, Rockford,USA) according to the manufacturer’s instructions with a peroxidase-conjugated anti-mouse antibody. The housekeeping gene β-actin was used as an internal control. The data were expressed as percentage to β-actin.
5
Dual Luciferase Assay of Transfected Cells
6
Screening of HIV-1 Inhibitors using Pseudotyped Viruses
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The metabolites M1, M2-9, M3-1, M3-2, M5-2, M5-3, M5-4, and M5-5 were screened for their activities against HIV-1 using pseudotyped viruses as described previously (Ma et al., 2013 (link)). Briefly, VSV-G/HIV-1 viruses were prepared by co-transfecting 293T cells with VSV-G plasmid and env-deficient HIV-1 vector (pNL4-3.luc.R-E-, Supplemental Figures S1 , S2 ). The virions were quantified by measuring the p24 levels using an enzyme-linked immunosorbent assay (ELISA) kit (ZeptoMetrix, Cat. 0801111) with a dilution of 0.2 ng p24/mL.
One day before infection, 293T cells were seeded at a density of 6 × 104 cells/well in 24-well plates and then infected with VSV-G/HIV-1 viruses in the presence or absence of the tested compounds. The infected cells were lysed 48 h post-infection using a cell lysis reagent (Promega), and the luciferase activity of the cell lysate was measured with a Sirius luminometer (Berthold Detection System). For comparison, the anti-HIV-1 activity of F18 was evaluated under the same conditions.
One day before infection, 293T cells were seeded at a density of 6 × 104 cells/well in 24-well plates and then infected with VSV-G/HIV-1 viruses in the presence or absence of the tested compounds. The infected cells were lysed 48 h post-infection using a cell lysis reagent (Promega), and the luciferase activity of the cell lysate was measured with a Sirius luminometer (Berthold Detection System). For comparison, the anti-HIV-1 activity of F18 was evaluated under the same conditions.
7
Alkaline Phosphatase Activity Assay
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The alkaline phosphatase (ALP) activities were examined using the modified Great Escape SEAP chemiluminescence assay (BD Clontech) and/or histochemical staining, as described before [16 (link),37 (link)]. For ALP histochemical staining, iMEF cells were induced for osteogenic differentiation using AdBMP9, and AdGFP was used as control. At indicated time points, cells were first fixed with 0.05% glutaraldehyde at room temperature for 10 min, then washed with distilled water, and finally subjected to histochemical staining with a mixture containing 0.1 mg/ml of napthol AS-MX phosphate and 0.6 mg/ml of Fast Blue BB salt and stored in the dark for 10 min. Histochemical staining was recorded with the use of bright light microscopy.
For the chemiluminescence assays, iMEFs were first lysed by cell lysis reagent (Promega, U.S.A.), and then 5 μl of the cell lysis liquid, 5 μl substrate (BD Clontech), and 15 μl Lupo buffer were fully mixed and stored in the dark at room temperature for 20 min. ALP activities were normalized by total cellular protein concentrations in each sample. Each assay condition was performed in triplicate, and the results were repeated in three independent experiments.
For the chemiluminescence assays, iMEFs were first lysed by cell lysis reagent (Promega, U.S.A.), and then 5 μl of the cell lysis liquid, 5 μl substrate (BD Clontech), and 15 μl Lupo buffer were fully mixed and stored in the dark at room temperature for 20 min. ALP activities were normalized by total cellular protein concentrations in each sample. Each assay condition was performed in triplicate, and the results were repeated in three independent experiments.
8
Transfection and Silencing Assays in Cell Lines
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HEK293T cells were co-transfected with reporter vectors along with other expression vectors or molecular clones using Lipofectamine 2000 (Invitrogen, USA) and harvested 36 h post-transfection for luciferase assay. The cells were lysed in cell lysis reagent (Promega, USA) and luciferase assays were performed using Steady-Glo substrate (Promega, USA) as described earlier (30 (link)). Jurkat cells were transfected using x-treme gene HP DNA transfection reagent (Roche Applied Bioscience, Germany).
For silencing studies, cells were first transfected with siRNA using Lipofectamine 2000 as per the manufacturer's instructions, followed by second transfection (reporter plasmid/ expression vectors) or infection as described earlier (52 (link)). Cells were harvested 48 h post-transfection/infection. Knockdown was confirmed by immunoblotting with respective antibodies. Immunoblotting with GAPDH served as the loading control.
For silencing studies, cells were first transfected with siRNA using Lipofectamine 2000 as per the manufacturer's instructions, followed by second transfection (reporter plasmid/ expression vectors) or infection as described earlier (52 (link)). Cells were harvested 48 h post-transfection/infection. Knockdown was confirmed by immunoblotting with respective antibodies. Immunoblotting with GAPDH served as the loading control.
9
Cardioprotective Effects of Mul on Doxorubicin-Induced Toxicity
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H9c2 cardiomyocytes were purchased from the American Type Culture Collection (ATCC) and cultured in DMEM supplemented with 10% FBS. Only H9c2 cells at 3-5 passages were used for this experiment. At approximately 75% confluence, these cells were starved with a serum-free DMEM medium for 24 hours. After that, H9c2 cardiomyocytes were incubated in DMEM medium containing PBS, DOX (1 μM), DOX (1 μM) plus 25 μM Mul, DOX (1 μM) plus 50 μM Mul, and DOX (1 μM) plus 100 μM Mul. Cells were harvested at 0 h, 12 h, 24 h, and 48 h. Cell viability was examined using the Cell Counting Kit-8 assay (CCK-8, #HY-K0301, MedChemExpress) according to the manufacturer's instructions. To inhibit the activation of AKT, H9c2 cardiomyocytes were pretreated with a specific AKT inhibitor (1 μM) for 24 h.
H9c2 cardiomyocytes were cultured in a six-well plate. After serum starvation for 24 h, H9c2 cells were electrotransfected with nuclear factor kappa-B- (NF-κB-) luc (0.03 μg) with a Neon® Transfection System (pulse width: 20 ms, pulse voltage: 1700 V). After that, these cells were treated with DOX and Mul (100 μM) for 48 h. At the end of this experiment, cells were lysed in 100 μl of a cell lysis reagent (Promega, Madison, USA). Luciferase activity was detected with a Promega Luciferase assay reagent (#E1500).
H9c2 cardiomyocytes were cultured in a six-well plate. After serum starvation for 24 h, H9c2 cells were electrotransfected with nuclear factor kappa-B- (NF-κB-) luc (0.03 μg) with a Neon® Transfection System (pulse width: 20 ms, pulse voltage: 1700 V). After that, these cells were treated with DOX and Mul (100 μM) for 48 h. At the end of this experiment, cells were lysed in 100 μl of a cell lysis reagent (Promega, Madison, USA). Luciferase activity was detected with a Promega Luciferase assay reagent (#E1500).
10
Quantifying Cellular Copper Uptake
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