The following antibodies were used for the detection of hematopoietic subpopulations: Gr1-Vioblue (clone RB6–8C5), CD11b-APC (clone M1/70), CD45.1-PE-Cy7 (clone A20), CD45.2-PerCP-Cy5.5 (clone 104), CD3e-V500 (clone 145–2C11 or 500A2), B220-PE (clone RA3–6B2), Sca1-PE-Cy7 (clone D7) and cKit-APC (clone 2B8). Data acquisition was performed on a FACSCanto II flow cytometer and analyzed using the FACSDiva 6.0 software (all Beckton & Dickinson). Dead cells were excluded by staining with eFluor780 (eBioscience, San Diego, CA, USA). Sorting of cells for subsequent genomic DNA (gDNA) isolation was performed on a FACSAria II flow cytometer (Beckton & Dickinson) running with FACSDiva 6.0 software. For flow cytometric analysis of murine and human PSCs cells the following antibodies were used: mSSEA1-APC, mCD41-APC, hTra-1–60-PE, hCD45-APC, hCD11b-APC; isotype-controls: mouse-IgG1a- APC, mouse-IgM-PE (all eBioscience). Data acquisition was performed on a FACScalibur (Beckton & Dickinson) and raw data were analyzed using the software FlowJo (TreeStar, Ashland, OR, USA).
Facsdiva 6
Manufactured by BD
Sourced in United States, Germany
The FACSDiva 6.0 software is a flow cytometry analysis software developed by BD (Becton, Dickinson and Company). It is designed to provide users with tools for data acquisition, analysis, and visualization of flow cytometry experiments. The software supports a wide range of BD flow cytometry instruments and enables users to perform advanced data analysis and reporting.
Automatically generated - may contain errors
Lab products found in correlation
Facscanto 2, by BD (38 mentions)
Facscanto 2 flow cytometer, by BD (28 mentions)
Lsrii flow cytometer, by BD (22 mentions)
Lsrfortessa, by BD (17 mentions)
Facsaria 2, by BD (15 mentions)
Facscalibur, by BD (13 mentions)
Facscanto 2 cytometer, by BD (13 mentions)
Lsrfortessa flow cytometer, by BD (12 mentions)
Lsr 2 flow cytometer, by BD (11 mentions)
Lsr 2, by BD (10 mentions)
Lsrii cytometer, by BD (8 mentions)
Sorp facsaria2, by BD (8 mentions)
Facscanto, by BD (7 mentions)
Cytofix cytoperm, by BD (7 mentions)
Facsaria flow cytometer, by BD (7 mentions)
Perm wash buffer, by BD (6 mentions)
Facsaria, by BD (6 mentions)
Propidium iodide, by Merck Group (6 mentions)
Human fcr blocking reagent, by Miltenyi Biotec (5 mentions)
Facsaria 3, by BD (5 mentions)
388 protocols using facsdiva 6
1
Immunophenotyping of Murine and Human PSCs
2
Apoptosis Detection: Vibrant FAM and Annexin V/PI
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Vibrant FAM polycaspase assay (V35117, Molecular Probes) was used as recommended by the manufacturer. Briefly, cells were harvested and incubated with the kit reagent solution for 1 hr at 37°C. After having been washed with the wash buffer provided in the kit, cells were incubated with propidium iodide (PI) (1 µg/mL) for 15 min on ice. Then, cells were analyzed by flow cytometry on a BD FACSCanto II (Becton Dickinson). Data analyses were made using the FACSDiva 6.0 software (BD Biosciences).
The Annexin V/PI assay (V13241, Molecular Probe) was used as recommended by the manufacturer. Briefly, cells were harvested, incubated with Alexa Fluor488-Annexin V and PI (1 µg/mL) for 15 min at room temperature. Then, cells were diluted in the buffer provided by the kit and analyzed by flow cytometry on a BD FACSCanto II (Becton Dickinson). Data analyses were made using the FACSDiva 6.0 software (BD Biosciences).
The Annexin V/PI assay (V13241, Molecular Probe) was used as recommended by the manufacturer. Briefly, cells were harvested, incubated with Alexa Fluor488-Annexin V and PI (1 µg/mL) for 15 min at room temperature. Then, cells were diluted in the buffer provided by the kit and analyzed by flow cytometry on a BD FACSCanto II (Becton Dickinson). Data analyses were made using the FACSDiva 6.0 software (BD Biosciences).
3
Cellular Immunity Assessment in EBRT
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To assess the dynamics of the IS, all patients underwent venous blood sampling before and after adjuvant EBRT 27 ± 3 days after the completion of surgical/chemotherapeutic treatment. Cellular immunity parameters were analyzed on the day of blood sampling by determining the absolute number and percentage of lymphocyte subpopulations with a flow cytometer (BD FACS Canto II, Becton Dickinson and Company, Franklin Lakes, NJ, USA), using a single-platform method with a commercial kit of monoclonal antibodies (Multitest IMK kit with BD Trucount, Becton Dickinson tubes) according to the manufacturer’s instructions. The results were evaluated using BD FACS Diva 6.0 software.
Normal indicators accepted in our laboratory of cellular immunity are shown inTable 1 .
The obtained data were statistically processed using the Statistica 6.0 software. The data between groups were compared using the two-tailed Fisher’s exact test. The mean values of the immune status parameters were expressed as means ± SEM and compared using the unpaired Student’s t-test; the paired t-test was used to analyze the dynamics of the parameters. For all criteria, the differences were considered statistically significant at p < 0.05.
Normal indicators accepted in our laboratory of cellular immunity are shown in
The obtained data were statistically processed using the Statistica 6.0 software. The data between groups were compared using the two-tailed Fisher’s exact test. The mean values of the immune status parameters were expressed as means ± SEM and compared using the unpaired Student’s t-test; the paired t-test was used to analyze the dynamics of the parameters. For all criteria, the differences were considered statistically significant at p < 0.05.
4
Flow Cytometry Analysis of MSC Viability
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After preactivation, the MSCs were trypsinized and seeded in six-well dishes at 5 × 105 cells per well. After 24 and 96 h, the MSCs were incubated with 50 μg/mL propidium iodide in PBS for five minutes. Flow cytometry was used to determine the percentage of dead cells. The data was analyzed in BD FACSDiva 6.0 software.
5
Apoptosis Analysis of MCF-7 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The MCF-7 cells were seeded at a density of 5 × 105 cells/ml in a Corning® 24-well plate. After 24 h incubation, cells were treated with either the half-maximal inhibitory concentration on cell viability (IC50) or 400 μg/ml of methanolic (MSD, MST, ML) or aqueous extracts (ASD, AST, AL) of the plant parts for an additional 24 h incubation. The cells exposed to 1 µM of staurosporine (STS) (Santa Cruz biotechnology, Dallas, TX, USA) were the positive control for the induction of apoptosis. The untreated cells, the control and unstained cells, were also used in the apoptotic status analysis. The cells were stained with fluorescein isothiocyanate (FITC)‐labeled Annexin V and phycoerythrin (PE)-labeled propidium iodide (PI), according to the manufacturer’s instructions for the FITC‐Annexin V Apoptosis Detection Kit I (Becton Disckinson Biosciences, San Diego, CA, USA). The apoptotic status of 10,000 cells were analyzed with the Becton Dickinson (BD) FACSCanto™ flow cytometer using BD FACSDiva™ 6.0 software (BD Biosciences), and characterized by Annexin V+ve/PI−ve for early apoptosis and by Annexin V+ve/PI+ve for late apoptosis. Viable and necrotic cells were also detected as characterized by Annexin V−ve/PI−ve and by Annexin V−ve/ PI+ve, respectively.
6
Quantifying Apoptosis Pathways in Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The shrinkage of cells was observed using a ZEISS inverted phase-contrast microscope (Peine, Germany). The activity of pro-apoptotic executioner caspase-3/7 and of the mitochondrial permeability transition pore opening in the untreated and treated cells were assessed using Image-iT™ LIVE Red caspase-3/7 detection kit and Image-IT™ LIVE Mitochondrial Transition Pore Assay Kit (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA), respectively, according to the manufacturer’s instructions and as previously described in [67 (link)]. The cells with activated caspase-3/7 (red fluorescence) and apoptotic cells (whole green fluorescence) with mitochondrial outer membrane permeabilization were observed under the confocal microscopy. The abnormal phosphatidylserine exposure was determined using the Becton Dickinson (BD) Annexin V apoptosis detection kit (Becton Dickinson, San Jose, CA, USA) as described in [68 (link)]. The apoptotic status of 10,000 cells based on the dual staining of Annexin V conjugated to FITC and PI conjugated to PE was analyzed using BD FACSDiva™ 6.0 software on the BD FACSCanto™ II flow cytometer.
7
Hematopoietic Stem Cell Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
MNCs from BM or FL were pre-incubated with biotin-conjugated anti-CD3e, anti-CD45R/B220, anti-Gr-1, anti-CD11b, and anti-Ter-119 antibodies, with anti-CD16/32 antibody to block the Fcγ receptors (CD11b antibody was excluded in FL MNCs)43 (link)–45 (link). The cells were then stained with streptavidin-fluorescein isothiocyanate (FITC) and anti-Sca-1-PE-Cy7, c-Kit-APC-Cy7, CD150-APC, and CD48-Pacific Blue antibodies. The frequencies of HPCs (Lin−/Sca-1−/c-kit+), LSK cells (Lin−/Sca-1+/c-kit+), and HSCs (CD150+/CD48−/LSK) were analyzed using an Aria II cell sorter. For each sample, approximately 5 × 105 to 1 × 106 cells were acquired and the data were analyzed using the BD FACSDiva 6.0 software (BD Biosciences) and the FlowJo software (FlowJo, Ashland, OR, USA) . The number of the different hematopoietic cell populations in each mouse was calculated by multiplying the total number of MNCs harvested from each mouse or embryo with the frequencies of each population of MNCs. The information for all antibodies used in the staining is provided in Supplementary Table 1 .
8
Immunophenotypic Analysis of Leukemia Cells
9
Murine Neutrophil Purity Validation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The purity of the murine neutrophil preparations was routinely confirmed via flow cytometry (LSRII, BD Technologies). Anti-Gr-1 antibody (Miltenyi Biotec, San Diego, CA, USA) was used against Gr-1-expressing granulocytes following the manufacturer’s recommendations. Cells were analyzed in BD LSRII flow cytometer (BD Biosciences, San Jose, CA, USA) using BD FACSDiva 6.0 software (BD Biosciences, San Jose, CA USA) at the Imaging Core Facility of the Department of Infectious Diseases at UGA. The described protocol resulted in more than 95% PMNs (S5 Fig ).
10
Stimulation and Flow Cytometry Profiling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Single suspension extracted cells, as described above, were plated into flow cytometry tubes (Sarstedt) at a concentration of 1 × 106 per ml. Cells were stimulated with 50 ng/ml phorbol 12-myristate 13-acetate (PMA), 1 μg/ml ionomycin, 2 μM monensin (all Sigma Aldrich) for 3–4 h as indicated. (Sigma Aldrich) FcR receptor-blocking antibodies were added before staining with antibodies. Surface staining antibodies were added with live/dead stain (Invitrogen). For intracellular staining, cells were fixed and permeabilised using the Foxp3 fixation/permeabilization buffer kit (Thermo Fisher) according to the manufacturer’s instructions. Samples were acquired using a BD LSRFortessa (BD Biosciences). Sample data was recorded in FCS 3.0 data format using BD FACSDiva 6.0 software (BD Biosciences). Analysis of the data was performed using FlowJo software (Treestar Inc., Ashland, OR, USA). Supplementary Table 3 provides the antibodies used and their dilutions.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!