FACS was used to analyze the expression levels of the various antigen markers of cultured DPSCs49 (link). SCs (1 × 106) were incubated with 1 μL of rat monoclonal antibodies against CD90, CD29, CD45, and CD34 (BD Bioscience, San Jose, CA; 1 μL) in 2% FBS at 4 °C for 30 min. Cells from the negative control group were incubated in the buffer without any major antibodies. The fluorescence signals were analyzed using FACSCalibur Cell Quest software (Becton Dickinson, USA).
Cell quest software facscalibur
Manufactured by BD
Sourced in United States
The Cell Quest software is a data analysis and management application designed for the FACSCalibur flow cytometry system. It provides tools for acquiring, analyzing, and managing flow cytometry data.
Automatically generated - may contain errors
5 protocols using cell quest software facscalibur
1
Characterization of DPSC Antigen Profiles
2
Evaluating Cell Membrane Integrity with Propidium Iodide
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Cells were incubated with the DNA-binding probe propidium iodide (PI), which penetrates cells only when membranes are damaged. Stock solutions of PI (Molecular Probes, Leiden, Netherlands) were prepared in distilled water to a final concentration of 10 g l À1 and stored in the dark at 4°C. PI was added to a final concentration of 0Á5 g l À1 and incubated for 5 min at room temperature. For the flow cytometry analysis, the cell concentration in the samples was adjusted to approx. 10 6 CFU ml À1 . PI uptake was followed by flow cytometry (FACSCALIBUR, CELL- QUEST software; Becton Dickinson, Mountain View, CA), according to a previously reported procedure (Hugo et al. 2012) . Samples were analysed before and after the freeze-drying process, as well as after incubation in synthetic wine at 21°C for 24 h. For each sample, 10 000 events were collected, keeping the event rate below 300 events s À1 . Untreated stained cells were used as negative control and cells heated for 3 min at 80°C as positive control.
3
Multicolor Flow Cytometry Analysis
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Camel or human cells (PBMC or leucocytes; 4 x 105) were incubated with mAbs specific for human CD antigens (Table 1 ) in PBS containing bovine serum albumin (5 g/l) and NaN3 (0.1 g/l). After 30 minutes incubation (4°C), cells were washed twice and analyzed on the flow cytometer. A Becton Dickinson FACSCalibur equipped with Cell Quest software (FACSCalibur (Becton Dickinson Biosciences, San Jose, California, USA) was used to collect the data. At least 100 000 cells were collected and analyzed with the FCS Express software Version 3 (De Novo Software, Thornton, Ontario).
In order to exclude signals due to non-specific binding of mouse antibodies, negative isotype controls for mouse IgG1, IgG2a, IgG2b (from BD) and IgM (from Beckmann Coulter) were also included as part of the study.
In order to exclude signals due to non-specific binding of mouse antibodies, negative isotype controls for mouse IgG1, IgG2a, IgG2b (from BD) and IgM (from Beckmann Coulter) were also included as part of the study.
4
Cell Cycle and Apoptosis Analysis in Lymphoma Cells
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Cell cycle analysis and apoptosis detection were performed after treating KM-H2 and L428 cells with celastrol at the described doses for 24 h. Cell cycle analysis was carried out by propidium iodide (PI) staining. Briefly, 2 × 105 cells were fixed with ethanol 70% and incubated in 20 µg/mL PI and 0.2 mg/mL RNAse A for 30 min at room temperature. Apoptosis was detected by Annexin/PI assays. After being washed twice with cold PBS, the cells were incubated with 100 µL of binding buffer containing 5 µL Annexin V-FITC (recombinant annexin V conjugated to green-fluorescent FITC dye) and 10 µL PI (20 µg/mL). The samples were analyzed in a FACSCalibur Flow Cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) collecting 10,000 events per analysis. DNA histograms and Annexin/PI dotplots were analyzed in CELLQuest software (FACSCalibur, version 5.1, Becton Dickinson, Franklin Lakes, NJ, USA).
5
Immunophenotyping of Leukocytes in Blood
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For the immunophenotyping of blood leukocytes [25 (link)], separated leukocytes were incubated with mouse monoclonal antibodies to the surface antigens CD4, WC1, and MHC-II and the cell adhesion molecules CD11a, CD11b, and CD18 diluted in MIF buffer for 15 min at 4°C. After incubation, cells were washed 3 times with MIF buffer by centrifugation at 300× g for 3 min and discarded the supernatant. Unlabeled primary antibodies were detected using fluorochrome-labeled secondary antibodies, and labeled cells were then analyzed by flow cytometry. A Becton Dickinson FACSCalibur equipped with Cell Quest software (FACSCalibur; Becton Dickinson Biosciences, San Jose, California, USA) was used to collect the data. At least 100,000 cells were collected and analyzed with the software FlowJo version 10 (Flowjo LLC, USA). Negative isotype controls for mouse IgG1, IgG2a, IgG2b (Becton Dickinson), and IgM (Beckman Coulter, CA, USA). were included as part of the study.
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