MCF-7/S cells were transfected with miR-16 inhibitor or mi-iNC, and MCF-7/A cells were transfected with miR-16 mimics or mi-mNC, as aforementioned. After 48 h, cells were treated with 10 µM ADM for 48 h to induce apoptosis. The attached and floating cells were harvested, and flow cytometry analysis was performed using an Annexin V-FITC/propidium iodide staining kit (Dojindo Molecular Technologies, Inc.) according to the manufacturer's protocol. Apoptotic cells were detected using a BD Accuri C6 Plus flow cytometer (BD Biosciences) and the data were analyzed by BD CSampler analysis software (cat. no. 653123; version 1.0.23.1; BD Biosciences). The percentage of apoptotic cells was calculated by dividing the number of proapoptotic and apoptotic cells by the total number of cells.
Csampler analysis software
Manufactured by BD
Sourced in United States
BD CSampler analysis software is a data analysis tool designed for use with BD instruments. The software enables users to capture, analyze, and report data generated by BD laboratory equipment.
Automatically generated - may contain errors
Lab products found in correlation
Accuri c6 plus flow cytometer, by BD (1 mentions)
Aldefluor, by STEMCELL (1 mentions)
Foxp3 staining kit, by Thermo Fisher Scientific (1 mentions)
Accuri c6 cell analyzer, by BD (1 mentions)
Lysotracker green dnd 26, by Thermo Fisher Scientific (1 mentions)
Tryple, by Thermo Fisher Scientific (1 mentions)
Rnase a, by Merck Group (1 mentions)
Gallios flow cytometer, by Beckman Coulter (1 mentions)
Percp cy5.5 anti mouse cd4, by BD (1 mentions)
Il 17a, by BD (1 mentions)
Percp cy5, by BioLegend (1 mentions)
Accuri c6, by BD (1 mentions)
Facs lysing solution, by BD (1 mentions)
5 protocols using csampler analysis software
1
Evaluating Apoptosis in Breast Cancer Cells
2
Quantifying ALDH-Positive Tumor Cells
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The p53 SMWC-treated and untreated human and mouse tumor cell lines were analyzed for ALDHpositive/ALDHbrightcellsby flow cytometry using ALDEFLUOR (StemCell Technologies Vancouver, BC, V5Z 1B3, Canada), as previously described [18 (link), 19 (link), 44 (link)]. In general, duplicate aliquots of 2 × 105 tumor cell samples were incubated with ALDEFLUOR, with or without the ALDH inhibitor, diethylaminobenzaldehyde (DEAB) (control), according to the manufacturer's instructions. The control aliquot was analyzed by flow cytometry and set for detection of ≤0.5% ALDHpositive cells. Using this cutoff, the test aliquot was analyzed to identify its ALDHpositive/ALDHbright cell content with ALDHbright cells defined as the ALDHpositive cells with double the mean fluorescence intensity (MFI) of the bulk population of ALDHpositive cells in a sample [19 (link)]. The flow cytometry analyses were performed using a C6-Sample cytometer (BD); all samples were run using identical settings to collect a minimum of 8,000-gated events. Analyses were done using BD CSAMPLER™ ANALYSIS software (BD). The effects of the p53 SMWC on cell growth and percentage content of ALDHpositive/ALDHbright cells were calculated relative to the untreated control cell populations.
3
Cell Cycle and Lysosomal Analysis
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For cell cycle analysis, 106 cells were fixed using the FoxP3 Staining Kit (00-5523-00 eBioscience) for 30 min at room temperature in the dark. Samples were then resuspended in permeabilization buffer containing 20 μg of RNase A (R6513 Sigma) and 1 μg of propidium iodide (PI) (130-093-233 Miltenyi Biotech) for 30 min. PI fluorescence was analyzed using a BD Accuri C6 cell analyzer with BD CSampler Analysis Software. Results were analyzed with FlowJo software version 10 (TreeStar).
For lysosomal analysis, confluent cells were incubated with 1 µM Lysotracker Green DND 26 (L7526-Thermo Fischer) diluted in complete RPMI medium for 30 min at 37°C. Cells were then detached by addition of TrypLE (12604021, ThermoFischer), washed in PBS 2% (v/v) FCS, and stained with 0.1 µM DAPI in PBS 2% (v/v) FCS immediately before analysis. Samples were processed on a Gallios Flow Cytometer (Beckman Coulter). Dead cells and doublets were excluded from analysis respectively by the selection of DAPI negative cells and co-analysis of integral vs time-of-flight side scatter signals. Data were analyzed on FlowJo software (BD Bioscience). Mean Fluorescence intensities (MFI) of lysotracker in each condition were normalized by performing a ratio with MFI of an unstained condition in the same channel.
For lysosomal analysis, confluent cells were incubated with 1 µM Lysotracker Green DND 26 (L7526-Thermo Fischer) diluted in complete RPMI medium for 30 min at 37°C. Cells were then detached by addition of TrypLE (12604021, ThermoFischer), washed in PBS 2% (v/v) FCS, and stained with 0.1 µM DAPI in PBS 2% (v/v) FCS immediately before analysis. Samples were processed on a Gallios Flow Cytometer (Beckman Coulter). Dead cells and doublets were excluded from analysis respectively by the selection of DAPI negative cells and co-analysis of integral vs time-of-flight side scatter signals. Data were analyzed on FlowJo software (BD Bioscience). Mean Fluorescence intensities (MFI) of lysotracker in each condition were normalized by performing a ratio with MFI of an unstained condition in the same channel.
4
Murine Splenic Immune Cell Analysis
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Individual mouse spleens were reduced to homogeneous single cells using 70 m nylon cell strainers (Corning, Tewksbury, MA, USA). Red blood cells (RBC) were processed three times or fewer using RBC lysis solution (Gibco, Grand Island, NY, USA) to count single cells. Finally, the cells were stained with PerCP-Cy5.5 Anti-Mouse CD4 (BD Biosciences, CA, USA), PE-conjugated Th1 (IFN-γ), or PE-conjugated Th17 (IL-17A) (BioLegend, San Diego, CA, USA). The fluorescence intensity was measured using a BD Accuri™ C5 flow cytometer system, and the data were processed using BD CSampler analysis software to determine the proportions of CD4+ and gated IFN-+ or IL-17A+ populations.
5
Characterizing Immune Cell Markers by Flow Cytometry
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Flow cytometry experiments were performed to characterize the expression of AnxA1, FPR1, FPR2, CD66b, Ly6G, and F4/80. To evaluate the expression of AnxA1 and CD66b in human neutrophils, isolated cells were fixed with FACS lysing solution (#349202 BD Biosciences), washed with PBS containing 0.1M glycine (Synth), permeabilized with 0.01% Triton-X-100, washed with 1% BSA in PBS (BSA/PBS), and incubated with primary anti-rabbit antibody to AnxA1 (1:100) for 1 h at 37 °C. Next, cells were washed with BSA/PBS and incubated with secondary goat anti-rabbit antibody conjugated to Alexa Fluor 488 (1:250; Invitrogen) and anti-PerCP-Cy™5.5 anti-human CD66b-neutrophil marker (1:50; #562254 BD Pharmigen) for 40 min in the dark at room temperature. To verify the frequency of mice circulating neutrophils (Ly6G+) and monocytes (F4/80+), cells were fixed with FACS lysing solution for 30 min at room temperature, washed with PBS containing 0.1M glycine, and incubated with anti-Ly6G conjugated with FITC (1:50; #551460 BD Pharmigen) and anti-F4/80 conjugated with PerCP-Cy5.5 (1:200; # 15-4801-82 Biolegend, San Diego, CA, USA) for 40 min in the dark at room temperature. The cells were analyzed by a flow cytometer (BD Accuri C6), taking 10,000 events into consideration and using BD CSampler™ Analysis software (version: 1.0.2641-21 BD Biosciences, Franklin Lakes, NJ, USA).
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