The effect of compound 6 on the cell cycle profile of cancer cells was determined according to a previous protocol (17 (link)). Briefly, cells were seeded at 3–4×105 cells per 6-cm dish and allowed to settle for 24 h. Log-phase cultures were exposed to compounds 5–7 (0 to 100 µM) or vehicle for 48 h. Cells were trypsinized, washed with PBS and fixed overnight in 95% ethanol at 4°C, and subjected to RNase treatment and stained for 30 min at room temperature with propidium iodide (PI; 500 µg/ml; Sigma-Aldrich; Merck KGaA). Cellular DNA content was determined by flow cytometry with a FACSCalibur flow cytometer with a 488 nm Coherent laser (BD Biosciences, Franklin Lakes, NJ, USA). The CellQuest Pro version 5.2.1 software (BD Biosciences) was used for data acquisition and analyses were performed using Modfit version 2.0 software (BD Biosciences).
Cellquest pro version 5
Manufactured by BD
Sourced in United States
CellQuest Pro Version 5.2.1 is a software application for data acquisition and analysis of flow cytometry experiments. It provides a user interface and tools for setting up, running, and analyzing flow cytometry experiments.
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Lab products found in correlation
Propidium iodide, by Merck Group (3 mentions)
Facscalibur, by BD (3 mentions)
Facscalibur flow, by BD (1 mentions)
Melon gel igg purification kit, by Thermo Fisher Scientific (1 mentions)
Neutravidin beads, by Thermo Fisher Scientific (1 mentions)
Aminolink plus immobilization kit, by Thermo Fisher Scientific (1 mentions)
Ecl protein biotinylation module, by GE Healthcare (1 mentions)
Facscanto 2 flow cytometer, by BD (1 mentions)
Cd4 pe, by Thermo Fisher Scientific (1 mentions)
Cd8 fitc, by Thermo Fisher Scientific (1 mentions)
B220 apc, by BD (1 mentions)
Gr1 fitc, by BD (1 mentions)
Dual laser facscan, by BD (1 mentions)
Sca 1 pe, by BD (1 mentions)
Ter119 fitc, by BD (1 mentions)
Mac1 pe, by BD (1 mentions)
Cd71 pe, by Thermo Fisher Scientific (1 mentions)
Annexin 5 apc, by BD (1 mentions)
Annexin 5 binding buffer, by BD (1 mentions)
Annexin 5 binding buffer, by Merck Group (1 mentions)
9 protocols using cellquest pro version 5
1
Cell Cycle Analysis of Cancer Cells Treated with Compound 6
2
Peptide Effects on Cell Membrane Integrity
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The effect of peptides on the cell-membrane integrity of E. coli BL21 and DH5α and lymphoma cells was studied using the flow cytometer analysis as described elsewhere53 (link). The cells were cultured and washed three times using PBS buffer and incubated for 30 minutes with peptides at different concentration. The incubated lymphoma cells were further processed referring to the annexin V-FITC apoptosis kit. Peptide treated and untreated cells (E. coli/lymphoma) were stained with 10 μg ml−1 propidium iodide (PI) and incubated for additional 30 min. The incubated cells were washed using PBS to remove unbound dye and flow cytometry analysis was carried out using the FACSCalibur and analyzed by CellQuest Pro Version 5.2.1 software (BD Biosciences, NJ, USA).
3
Phagocytosis Assay Using Biotinylated SE36
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SE36 was biotinylated by ECL Protein Biotinylation Module (GE healthcare) and then the biotinylated SE36 was coated on 1 μm yellow-green fluorescent (505/515 nm) neutravidin beads (Thermo Fisher Scientific). IgG was purified using the Melon Gel IgG Purification kit (Thermo Fisher Scientific) and re-suspended in PBS. The VTN-depleted sera were prepared by anti-VTN IgG-immobilized column using AminoLink Plus Immobilization Kit according to the manufacturer's instructions (Thermo Fisher Scientific). Phagocytosis assays were performed as previously described in ref.54 (link), with minor adjustments. In brief, 5 × 104 THP-1 cells were added to each well in a final volume of 100 μL, and the plate was incubated overnight under standard tissue culture conditions (37 °C, 5% CO2). After incubation, 5 × 105 beads were placed in each well of flat-bottom 96-well plates and incubated for 6 h. Cells were washed in ice-cold PBS by centrifugation at 500 g for 5 min at 4 °C. In each experiment, at least 10,000 events were recorded using FACSCalibur flow cytometer (FCM) and analysed using CellQuest Pro Version 5.2.1 software (BD Biosciences, Franklin, NJ, USA). The phagocytosis index (PI) (%) was calculated from the ratio of median fluorescent intensity (MFI) of THP-1 cells that engulfed tested beads/MFI of THP-1 cells that engulfed SE36-beads × 100.
4
Analyzing Mitochondrial Membrane Potential
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Rhodamine 123 is a mitochondrial-specific fluorescent probe that stains in a membrane potential-dependent fashion. This was used in the present study and was. The mitochondrial membrane potential (Δψmit) was analyzed with a rhodamine 123 assay purchased from Thermo Fisher Scientific, Inc, as previously described with minor modifications (13 (link)). Cells were trypsinized, and the cell suspension was transferred into a centrifuge tube for centrifugation (400 × g, 15 min, 37°C). The supernatant was aspirated, washed three times with PBS, and incubated with 5 µM rhodamine 123 at 37°C for 30 min in the dark. Rhodamine 123 fluorescence intensity was then detected with a BD-FACS Canto™ II flow cytometer (Becton-Dickinson) and CellQuest™ Pro version 5.2.1 (BD Biosciences). Experiments were repeated three times.
5
Multiparametric Flow Cytometry for Donor Chimerism
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To evaluate donor chimerism, PB samples were treated with a red blood cell (RBC) lysis buffer, and stained with CD45.2 (Ly5.2)-Cy5 (eBioscience) for S-HSCT or Ly9.1-FITC (BD Pharmingen) for A-HSCT, as follows. The cells were resuspended with Hank’s Balanced Salt Solution (HBSS) (Ca2+, Mg2+ free, Invitrogen, CA) containing 2% fetal bovine serum (FBS) (HF2 solution) and then incubated for 30 min on ice with fluorochrome-conjugated antibodies. Following staining, cells were washed twice with phosphate buffered saline (PBS) and then resuspended with HF2 containing 1ug/ml of propidium iodide (Sigma). The analyses were performed using a dual laser FACScan (BD Bioscience). Mice that were clinically ill were euthanized using CO2 narcosis. BM, spleen, and thymus were harvested and stained with antibodies to Mac-1-PE (BD Pharmingen), Gr-1-FITC (BD Pharmingen), B220-APC (BD Pharmingen), CD4-PE (eBioscience), CD8-FITC (eBioscience), CD71-PE (eBioscience), Ter119-FITC (BD Pharmingen), cKit-FITC (BD Pharmingen), Sca-1-PE (BD Pharmingen), and analyzed as described above. Cell quest pro version 5.2.1 (BD Bioscience) software was used for flow cytometry analysis.
6
Cell Cycle Analysis by Flow Cytometry
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The cell cycle was analyzed via flow cytometry. After 48 h post-transfection, HTR8/SVneo cells were trypsinized in chilled PBS, fixed in 70% ethanol for 24 h at −20°C, and then stained with propidium iodide for 10 min on ice. The samples were calculated using a flow cytometer and CellQuest Pro version 5.1 software (BD Biosciences).
7
Apoptosis Evaluation by Flow Cytometry
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Cell apoptosis was evaluated by flow cytometry (FCM), which was performed on a FACScan flow cytometer (BD Biosciences, San Jose, CA, USA). Briefly, Huh7 cells were treated with the three respective doses (140, 280 or 560 µM) of SIN or vehicle for at 37°C for 36 h. Following harvesting, the cells were washed three times with cold saline. Following centrifugation at 450 × g and 4°C for 10 min, the cell pellets were diluted with annexin V binding buffer (BD Biosciences) at 1×106 cells/ml. Then, 5 µl APC Annexin V (BD Biosciences) was added to 100 μl of the cell suspension, which was followed by incubation for 10 min at room temperature. The cells were washed and resuspended in 200 µl of the annexin V binding buffer, then stained with 5 μl propidium iodide (Sigma-Aldrich; Merck KGaA). CellQuest Pro version 5.1 (BD Biosciences) was used to analyze the data.
8
Apoptosis Analysis via Annexin V-FITC and 7-AAD
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Apoptosis was analyzed by the double-staining method using FITC-conjugated annexin V apoptosis detection kit and 7-amino-actinomycin D (7-AAD) purchased from BD PharmingenTM (BD Biosciences, San Jose, CA, USA) and Sigma-Aldrich, respectively. A total of 105 cells/well were cultured in a 6-well plate. Following a 24-h incubation at 37 °C, cells were treated with the considered concentration based on CI value and incubated for 48 h at 37 °C. Cells were collected and resuspended in 1× annexin binding buffer at a concentration of 1 × 105 cells/500 µL, followed by staining with annexin V-FITC or 7-AAD (BD 556570, BD Biosciences). Stained cells were analyzed using FACSCalibur (342973, BD FACSCalibur™, San Jose, CA, USA), and apoptotic cells were analyzed using Cell Quest Pro version 5.2 (BD Biosciences).
9
Annexin V/7-AAD Apoptosis Detection
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Apoptotic cell death was analyzed by Annexin V/7-AAD double staining. Cells were stained with Annexin V followed by staining with 7-AAD in the dark for 15 min at room temperature. Stained cells were detected by FACSCalibur (BD Biosciences, San Jose, CA, USA), and apoptotic cells were analyzed using CellQuest Pro version 5.2 (BD Biosciences, San Jose, CA, USA) software.
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