Cells were seeded at 1 × 106 per well in 12-well plates and treated with the lowest significant doses (LSDs) of apigenin (AP) (Sigma Aldrich, 10798) at 10 µM and 50 µM, etoposide (ETP) (Sigma Aldrich, E1383) at 0.01 µM and CYCLOphosphamide (CYCLO) (Sigma Aldrich, PHR1404) at 2 µM and 10 µM, alone or in combination; along with a vehicle control for 24 h. The therapeutic agents: AP, ETP, and CYCLO were prepared as described previously55 (link),63 (link),64 (link). LSDs capable of inducing apoptosis were selected based on our earlier published data55 (link),63 (link),64 (link) which caused a 10% to 20% increase in caspase-3 activity and induced apoptosis in THP-1 and Jurkat cells, when compared to the vehicle control.
Cyclo
Manufactured by Merck Group
Sourced in United States
The Cyclo is a piece of laboratory equipment designed for cell lysis and tissue homogenization. It utilizes a rotational mechanism to disrupt biological samples, allowing for the extraction and isolation of cellular components such as proteins, nucleic acids, and organelles. The Cyclo operates at consistent speeds and rotational forces to ensure efficient and reproducible sample preparation.
Automatically generated - may contain errors
Lab products found in correlation
Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions)
D cycloserine, by Merck Group (1 mentions)
L fucose, by Merck Group (1 mentions)
Ly294002 ly, by Merck Group (1 mentions)
Tnf α, by Thermo Fisher Scientific (1 mentions)
Staurosporine stauro, by Merck Group (1 mentions)
Lithium chloride (licl), by Carl Roth (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Wnt3a, by R&D Systems (1 mentions)
5 protocols using cyclo
1
Apoptosis Induction by Apigenin, Etoposide, and Cyclophosphamide
2
Evaluating Cytotoxic Activity of TsV
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The cytotoxic activity of TsV was evaluated using the MTT method described by Mosmann [18 (link)]. PBMC (5 × 105 cells/well) were cultured in 96-well plates in RPMI 1640 complete culture medium containing 10 % fetal calf serum (FCS) in the presence of TsV (12.5; 25.0; 50.0; 62.5; 100.0; 125.0; 250.0; 500.0; 1000.0; 2000.0 μg/mL) or Cyclophosphamide (Cyclo; 25 mg/mL; positive control; Sigma-Aldrich, USA) previously diluted in culture medium, for 24 h at 37 °C, and under 5 % CO2. Next, 20 μL of MTT (Sigma-Aldrich, USA) solution at 5 mg/mL was added to each well, and the plates were incubated for 4 h, at 37 °C, and under 5 % CO2. After incubation, the precipitated formazan crystals were dissolved with 200 μL of 20 % SDS (sodium dodecylsulfate), and absorbance was recorded at 570 nm. Absorbance values recorded for untreated cells (negative control) represent 100 % of PBMC viability, and were used as reference to calculate the percentage of cell viability in the presence of each sample concentration.
3
Pharmacological Modulation of Memory
4
LecB Regulation of Lung Cancer Cells
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H1299 and H1975 lung epithelial cells were grown at 37 °C and 5% CO2 in RPMI-1640 (Gibco) with 10% FCS and 2 mM l -glutamine (Gibco).
Lyophilized, recombinant LecB (Dr. Anne Imberty) was dissolved in PBS (with Ca/Mg) and used at concentrations of 0.09 μM, 0.9 μM and 4.3 μM for indicated time periods. Starvation in serum-free medium was performed, when indicated, for 2 h before treatment and maintained during stimulation. In order to block fucose-binding sites of LecB,l -fucose (Sigma-Aldrich) was directly dissolved in medium, sterile filtered and used at a final concentration of 43 mM for the same time periods as LecB. Other proteins for stimulation purposes were used at the following concentrations: Wnt3a (400 ng/ml, R&D Systems), TNF-α (10 ng/ml, Life Technologies) and staurosporine (Stauro, 1 μM, Sigma-Aldrich). The used inhibitors were pre-incubated for 30 min at 37 °C and maintained during stimulation at following concentrations: lactacystin (Lact, 10 μM, Biomol), bafilomycin (Baf, 0.2 μM, Invivogen), LY294002 (LY, 100 μM, Sigma-Aldrich), triciribine (TCN, 10 μM, Sigma-Aldrich), Akt1/2 inhibitor (Akti, 10 μM, Sigma-Aldrich), TWS119 (TWS, 20 μM, Selleckchem), lithium chloride (LiCl, 20 mM, Roth) and Cycloheximide (Cyclo, 100 μg/ml, Sigma-Aldrich).
Lyophilized, recombinant LecB (Dr. Anne Imberty) was dissolved in PBS (with Ca/Mg) and used at concentrations of 0.09 μM, 0.9 μM and 4.3 μM for indicated time periods. Starvation in serum-free medium was performed, when indicated, for 2 h before treatment and maintained during stimulation. In order to block fucose-binding sites of LecB,
5
Optimizing Cell Proliferation Inhibition
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Cells were seeded at 2.5x103 cells/well and incubated overnight. Cycloheximide (Cyclo, protein translation/proliferation inhibitor, Sigma) and Latrunculin A (Lat A, actin stabilizer, Calbiochem) were added at 0, 50, 100, 200, and 500 nM concentrations for Cyclo and 0, 1, 5, 10, and 20 nm concentrations for Lat A for an additional 24 h incubation. Thiazolyl Blue Tetrazolium Bromide MTT solution was then added for 3 h followed by lysis of the cells. Absorbance was read at 570nm. For Cyclo, a value of 50% compared to control was considered satisfactory for blocking most of the proliferative capacity. For Lat A, a % viability greater than 80% was deemed necessary to avoid the impact of cell death on the measurements.
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