For analysis of Tregs, mAbs with specificity against CD4 (RM4-4) and CD25 (7D4, non-cross-reactive with PC61) were used. Antibodies were conjugated to FITC and PE and detected in FL1 and FL2. Surface staining was performed according to standard procedures, and flow cytometric analysis was performed on a Coulter Cytomics FC500. CXP software (Coulter, Vienna, Austria) was used for acquisition and analysis.
Cxp software
Manufactured by Beckman Coulter
Sourced in United States, France, Germany, Canada
The CXP software is a data analysis and visualization tool designed for use with Beckman Coulter flow cytometry instruments. It provides users with the ability to acquire, analyze, and manage flow cytometry data.
Automatically generated - may contain errors
Lab products found in correlation
Cytomics fc500, by Beckman Coulter (56 mentions)
Fc500 flow cytometer, by Beckman Coulter (39 mentions)
Cytomics fc500 flow cytometer, by Beckman Coulter (23 mentions)
Fc500, by Beckman Coulter (23 mentions)
Fc500 cytometer, by Beckman Coulter (6 mentions)
Rnase a, by Merck Group (6 mentions)
Annexin 5 and pi staining kit, by BD (6 mentions)
Fc500 mpl cytometer, by Beckman Coulter (4 mentions)
Propidium iodide, by Merck Group (4 mentions)
Cytomics fc 500 series, by Beckman Coulter (4 mentions)
Fc500 flow cytometry system, by Beckman Coulter (4 mentions)
Fitc annexin 5 apoptosis detection kit 1, by BD (4 mentions)
Pma ionomycin, by Merck Group (3 mentions)
Kaluza analysis software 2, by Beckman Coulter (3 mentions)
Golgistop, by BD (3 mentions)
Hla dr, by BD (3 mentions)
Formaldehyde, by Thermo Fisher Scientific (2 mentions)
Flow cytometry, by Beckman Coulter (2 mentions)
Annexin 5 pi apoptosis detection kit, by BD (2 mentions)
Anti il 17a, by BioLegend (2 mentions)
238 protocols using cxp software
1
Flow Cytometric Analysis of Tregs
2
Platelet Surface Marker Expression Analysis
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Activated GPIIb/IIIa (JON/A) and CD62P (P-selectin) were used as markers of platelet activation and secretion, respectively. Platelet surface expression of selected markers was also evaluated such as GPIb α , GPIb β , GPIV (also known as CD36), GPVI, and integrin α 2 and α IIb chains.
Diluted whole blood or washed platelets (2.10 6 /mL) were stimulated with a range of agonists, as indicated. After incubation for 5 min at room temperature without stirring, whole blood or washed platelets were incubated with appropriate fluorophore-conjugated antibodies for 10 min at room temperature. The reaction was stopped by adding 500 L of PBS, and immediately analyzed with a FC500 flow cytometer (Beckman Coulter). The platelet population was gated using their forward and side scatter characteristics. Acquisition and processing data from 10,000 platelets were collected and analyzed using CXP software (Coulter). The Mean fluorescence intensity (MFI) of the whole platelet population was used.
Diluted whole blood or washed platelets (2.10 6 /mL) were stimulated with a range of agonists, as indicated. After incubation for 5 min at room temperature without stirring, whole blood or washed platelets were incubated with appropriate fluorophore-conjugated antibodies for 10 min at room temperature. The reaction was stopped by adding 500 L of PBS, and immediately analyzed with a FC500 flow cytometer (Beckman Coulter). The platelet population was gated using their forward and side scatter characteristics. Acquisition and processing data from 10,000 platelets were collected and analyzed using CXP software (Coulter). The Mean fluorescence intensity (MFI) of the whole platelet population was used.
3
Immunophenotyping of Mesenchymal Stem Cells
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Briefly, 1 × 105 cells at passage 3 or 4 were stained for 15 min in 100 μL phosphate-buffered saline with fluorescein isothiocyanate (FITC)- or phycoerythrin (PE)-conjugated antibodies to detect CD34, CD45, CD44, CD73, CD90, or CD105. Antibodies were sourced from BD Biosciences (Le Pont de Claix, France), except CD105 which was obtained from BioLegend through Ozyme (Paris, France). The ratio of mean fluorescent intensity (MFI) was defined as the ratio of MFI obtained with one antigen-surface marker to the MFI obtained with the corresponding isotype (IgG-FITC or IgG-PE). Fluorescent intensities of 50,000 events were acquired using a FC500 flow cytometer and CXP software (version 2.2, Beckman Coulter, Villepinte, France).
4
Retroviral Transduction Efficiency Assay
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The cells were seeded into 24-well plates at 2 × 104 cells/well (MEF) or 1 × 105 cells/well (SupT1) and infected the following day with GFP-expressing retroviral vectors. MEF cells were trypsinized at 2 dpi and fixed in 3 % formaldehyde (Fisher Scientific, MA, USA). The percentage of GFP-positive cells and MFI were then determined by analyzing 104 cells on a FC500 MPL cytometer (Beckman Coulter, CA, USA) using the CXP Software (Beckman Coulter). MFI analysis was restricted to the GFP-positive cells.
5
Quantifying HIV-1 Infection in CRFK Cells
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CRFK cells were plated in 24-well plates at 30,000 cells per well and infected the next day with different amounts of HIV-1-GFP or using a defined multiplicity of infection (MOI). Two days post-infection, cells were trypsinized and fixed in 2% formaldehyde in a PBS solution. The % of GFP-positive cells was then determined by analyzing 10,000 cells using a FC500 MPL cytometer with the CXP software (Beckman Coulter).
6
Apoptosis Evaluation of SK-N-MC Cells
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Annexin V and PI staining to evaluate apoptosis of SK-N-MC cells was performed by using an Annexin V/PI apoptosis detection kit (BD Bioscience, Franklin Lakes, NJ, USA) according to the manufacturer’s manual. Briefly, SK-N-MC culture medium was collected after cell treatment. The SK-N-MC cells were detached and suspended with 0.25% trypsin and a 0.5 mM EDTA solution. Next, the collected medium was added to the cell suspension. Suspended cells were counted by using a Petroff-Hausser counting chamber (Hausser Scientific). Cells (1 × 105) were then resuspended in binding buffer supplied with the apoptosis detection kit and immune-stained with 5 μl of FITC-Annexin V and 5 μl of PI for 15 min in the dark at room temperature. Apoptosis analysis was performed by using flow cytometry (Beckman Coulter). Cell (3 × 105) that had similar levels of side scatter value and cell volume were measured and analyzed by using CXP software (Beckman Coulter).
7
Quantifying Insulin Expression in iPSCs
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To determine the level of insulin expression in iPSCs after different treatments, the cells were analyzed using an anti-insulin antibody (1:200, Abcam, United States) in an FC500 flow cytometer (Beckman Coulter, United States). Briefly, cells were collected, blocked, and labeled with the FITC-conjugated antibody against insulin in accordance with the manufacturer’s instructions. The data were analyzed with CXP software (Beckman Coulter). iPSCs were used as a negative control. Mean fluorescence intensity was determined after subtracting the value of negative control (iPSCs).
8
Multiparametric flow cytometry of T-cell subsets
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We used flow cytometry analysis to assess the production of FOXP3, Helios, GATA3, IL-9, IL-17A, and IL-10 by CXCR6+ and CD4+ T cells. Briefly, splenocytes were incubated with phorbol 12-myristate 13-acetate (PMA)/ionomycin (Sigma-Aldrich) for 4 h in the presence of brefeldin-A (GolgiPlug, BD Biosciences), which prevents the transport of cytokines and transcription factors out of the cell [12 (link),33 (link),41 (link)]. Cells were washed and surface stained for CD4, and CXCR6 surface receptors (BioLegend, San Diego, CA, USA). After permeabilization and fixation (BioLegend), the cells were stained with intracellular cytokines (anti-IL-9, anti-IL-10, and anti-IL-17A; BioLegend) and transcription factors (anti-FOXP3, anti-GATA3, and anti-Helios; BioLegend). The proportions of CXCR6+FOXP3+, CXCR6+Helios+, CD4+GATA3+, CD4+IL-9+, CXCR6+IL-10+, and CD4+IL-17A+ cells were acquired via a FC 500 flow cytometer and analyzed using CXP software (Beckman Coulter, Indianapolis, IN, USA).
9
Ascorbate and Phosphoascorbate Effects on SKM-1 Cells
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SKM-1 cells were cultured for 6 days with the addition of sodium ascorbate, bovine liver catalase, and phosphoascorbate (2-phospho-L-ascorbic acid trisodium salt) where indicated. Cells were seeded at 0.2 x 106 cells/ml in a volume of 1 ml in a 24-well plate. Ascorbate and phosphoascorbate were added at 0 – 500 μM and catalase at 20 μg/ml. The media was refreshed with a 1:5 dilution at days 2 and 4, with the addition of ascorbate, catalase and phosphoascorbate at the original concentration. The number of cells was recorded after 6 days with a hemocytometer and viability assessed by trypan blue dye exclusion. PI/Annexin V and PI DNA staining were used to assess cell viability/apoptosis and cell cycle status via flow cytometry after 6 days with 300 μM ascorbate, with data analysis using CXP Software from Beckman Coulter (Brea, CA, USA).
10
Cell Cycle Analysis of Melanoma Cells
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518A2 melanoma cells (10 × 104 cells/mL, 3 mL/well) were cultivated in six-well plates for 24 h. After the treatment with test compounds (10 and 25 nm), C-A4 (10 and 25 nm), or DMSO (vehicle) for an additional 24 h, the cells were trypsinized, centrifuged (300 × g, 5 min, 4 °C), and fixed in 70% EtOH for min. 24 h. For FACS measurement, cells were washed in PBS and stained with propidium iodide solution (50 µg/mL PI, 50 µg/mL RNase A in 0.1% sodium citrate) for 30 min at 37 °C. The DNA content of at least 10000 single cells was determined using a Beckmann Coulter Cytomics FC500 flow cytometer (λex = 488 nm, λem = 570 nm). CXP software (Beckman Coulter) was used to analyze the cells in the cell cycle phases (sub-G1, G1, S, G2/M).
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