To quantify the cytokine levels, T-cells were isolated from the thymi of Lass4 WT and Lass4 LCK/Cre mice. Primary T-cells were differentiated into cytotoxic T-cells by adding 60 ng/mL human TGFβ in the presence of IL-2 and β-mercaptoethanol. For activation, the primary T-cells were incubated with CD2/3/28-activation beads for 4 h and 8 h. The supernatant of the cell culture was harvested and stained with a cytometric bead array flex set (mouse IL-6, IL-10, IL-17A, IFNγ and TNFα; BD Biosciences). Sample acquisition was performed with a LSR Fortessa flow cytometer (BD Bioscience) and analysis was conducted with BD Biosciences FCAP software (V3.0).
Fcap software v3.0
Manufactured by BD
FCAP software (V3.0) is a data analysis software for flow cytometry applications. It provides tools for processing and visualizing data generated from flow cytometry instruments.
Automatically generated - may contain errors
Lab products found in correlation
Accuri c6, by BD (4 mentions)
Accuri, by BD (4 mentions)
Lsrii fortessa flow cytometer, by BD (3 mentions)
Cytometric bead array flex set, by BD (3 mentions)
Lsrfortessa flow cytometer, by BD (2 mentions)
Cba cytokine inflammatory kit, by BD (2 mentions)
Transwell filters, by Greiner (1 mentions)
Cytometric bead array cba human th1 th2 th17 cytokine kit, by BD (1 mentions)
Cba flex set, by BD (1 mentions)
Complete edta free protease inhibitor cocktail, by Roche (1 mentions)
Accuri flow cytometer, by BD (1 mentions)
10 protocols using fcap software v3.0
1
Cytokine Profiling of Activated T-cells
2
Transwell Assay for Leukocyte Migration
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Conditioned basal RPMI medium (750 µl) was collected from WT macrophages 24 h after M1 or M2 polarization or from nonpolarized controls and added to the lower compartment of Transwell filters (3.0-µm cell culture inserts from Greiner Bio-one). Optionally, 50 ng/ml CCL2 was used as chemoattractant in some assays. BM-derived leukocytes isolated from WT and VASP-KO (5 × 105 cells/350 µl basal RPMI medium containing 0.1% BSA) were added into the upper chamber of Transwell filters and allowed to migrate for 2 h toward the chemoattractant.
To investigate the impact of Rac1, cells were preincubated with 10 µM of the specific Rac1 inhibitor Ehop-016 (Montalvo-Ortiz et al., 2012 (link)) for 1 h before migration. Migrated leukocytes were collected and identified by FACS using a LSRII/Fortessa flow cytometer, and results were evaluated with FCAP software v.3.0 (BD Biosciences). Neutrophils were defined as CD3− CD11b+ Ly6Ghigh Ly6Clow cells, and monocytes as CD3− CD11b+ Ly6Glow, Ly6Chigh cells.
To investigate the impact of Rac1, cells were preincubated with 10 µM of the specific Rac1 inhibitor Ehop-016 (Montalvo-Ortiz et al., 2012 (link)) for 1 h before migration. Migrated leukocytes were collected and identified by FACS using a LSRII/Fortessa flow cytometer, and results were evaluated with FCAP software v.3.0 (BD Biosciences). Neutrophils were defined as CD3− CD11b+ Ly6Ghigh Ly6Clow cells, and monocytes as CD3− CD11b+ Ly6Glow, Ly6Chigh cells.
3
Cytokine Profiling in Cell Culture
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To determine cytokine levels in cell culture supernatants human IL-1β, and TNF-α or murine IL-1β, IL-, IL-10, IL-23 (p19/p40), TNF-α and CCL5 Cytometric Bead Array Flex Sets (BD Biosciences, Franklin Lakes, NJ, USA) were used. Samples were acquired with a LSRII/Fortessa flow cytometer (BD Biosciences) and data were analyzed using BD Biosciences FCAP software (V3.0).
4
Murine Cytokine Profiling via CBA
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For determination of certain blood cytokine levels, as described before (23 (link)), murine IL-1β, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-17, TNF-α, IFN-γ, and MCP-1 Cytometric Bead Array flex sets (BD Biosciences) were used. Samples were acquired with a BD LSR II Fortessa flow cytometer (BD Biosciences) and data were analyzed with BD Biosciences' FCAP software (V3.0).
5
Salivary Cytokine Analysis in Cerebral Palsy
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For a subset of 37 participants, all of whom were CP subjects, we analyzed the salivary cytokines IL-1β, IL-6, IL-8, IL-10, and TNF-α. The analysis of cytokines in saliva was performed using a CBA Cytokine Inflammatory Kit (Becton Dickinson, CA, USA) for the detection of TNF-α, IL-1β, IL-6, IL-8, IL-10. All analyses were performed in duplicate. Briefly, 25μL of fluorescent particles conjugated to antibodies specific for each cytokine were added to 25 μL of the saliva and incubated for 1 h at room temperature away from light. Subsequently, 25 μL of the secondary antibody conjugated to a fluorochrome was added to the mixture and incubated for 2 h at room temperature. The results were compared to a standard curve with serially diluted cytokines. The particles were washed to remove unbound antibodies, resuspended in the wash buffer, and analyzed using a BD Accuri (BD Biosciences). Data acquisition was performed using BD-Accuri C6 Software, and concentrations were determined using FCAP software v.3.0 (BD Biosciences).
6
Cytokine Quantification in Polarized Macrophages
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Quantification of CXCL1 and CCL2 in cell culture supernatants and muscle lysates was performed by use of cytometric bead array flex sets (BD Biosciences). In brief, BM-derived macrophages from WT mice were polarized to M1 (10 ng/ml LPS + 1 ng/ml IFNγ) or M2 (25 ng/ml IL-4) or left untreated (control; CTL). 24 h later, the conditioned media were collected, and 25 µl of each sample was incubated with 25 µl CXCL1/CCL2-specific beads followed by CXCL1/CCL2-specific PE-labeled antibodies. After washing, samples were resuspended in 250 µl FACS Flow, measured on a LSRII/Fortessa flow cytometer, and evaluated with BD Bioscience FCAP software v.3.0.
7
Cytokine Profiling in Lymphocyte Cultures
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After the FA treatments, in the presence and absence of ConA for 24 h, the measurement of TNF-α, IL-6, IL-4, IL-2, IL-10, IFN-γ, and IL-17A concentrations in the lymphocyte culture supernatant was performed using the BD™ Cytometric Bead Array (CBA) Human Th1/Th2/Th17 Cytokine kit (BD Biosciences), according to manufacturer’s instructions. These cytokines are largely produced by Th1, Th2, and Th17 lymphocytes.
Briefly, 25 μL of particles containing different fluorescent beads and covered with specific antibodies for the cytokines were added to 25 μL of diluted culture supernatant and incubated for 1 h, at room temperature in the dark. Afterwards, 25 μL of the secondary antibody conjugated to a fluorochrome were added to the suspension, followed by the incubated for 2 h, at room temperature. At the same time, the standards for each cytokine were similarly used in the absence of the samples. The particles were washed to remove the unbound antibodies, resuspended in washing buffer and analyzed by using the BD Accuri (BD Biosciences). The acquisition was made in BD-Accuri C6 Software and the cytokine concentrations determined using the FCAP Software v.3.0 (BD, Biosciences).
Briefly, 25 μL of particles containing different fluorescent beads and covered with specific antibodies for the cytokines were added to 25 μL of diluted culture supernatant and incubated for 1 h, at room temperature in the dark. Afterwards, 25 μL of the secondary antibody conjugated to a fluorochrome were added to the suspension, followed by the incubated for 2 h, at room temperature. At the same time, the standards for each cytokine were similarly used in the absence of the samples. The particles were washed to remove the unbound antibodies, resuspended in washing buffer and analyzed by using the BD Accuri (BD Biosciences). The acquisition was made in BD-Accuri C6 Software and the cytokine concentrations determined using the FCAP Software v.3.0 (BD, Biosciences).
8
Cytokine Quantification in Murine Samples
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To determine cytokine levels in cell culture supernatants or murine skin (as described before [12 (link)]), murine IL-17A, IL-1β, IL-6, IL-23, CCL5, and CCL2 Cytometric Bead Array (CBA) Flex Sets (BD Biosciences) were used. Mouse skin tissue supernatant was prepared by homogenization of snap frozen tissue using ceramic pestle and mortar and extracted in modified RIPA buffer (50 mM Tris/HCl pH 7.5, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate, 150 mM NaCl, complete EDTA-free protease inhibitor cocktail (Roche, Mannheim, Germany), and 1 mM PMSF). Samples were acquired with an LSR Fortessa flow cytometer (BD Biosciences) and data was analyzed using the BD Biosciences FCAP software (V3.0).
9
Cytokine Profiling by Flow Cytometry
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TNF-alpha, IL-17, IL-4, IL-6, IL-2, IL-10, and IFN-gamma concentrations in the supernatant were determined by Cytometric Bead Array (CBA), using the BD™ CBA Th1Th2Th17 Human Cytokine Kit (BD Biosciences) and a BD Accuri flow cytometer. Briefly, 25 μL of particles containing different fluorescent beads and covered with specific antibodies for the cytokines were added to 25 μL of diluted culture supernatant and incubated for 1 h, at room temperature in the dark. Afterward, 25 μL of the secondary antibody conjugated to a fluorochrome was added to the suspension, followed by the incubated for 2-h, at room temperature. At the same time, the standards for each cytokine were similarly used in the absence of the samples. The particles were washed to remove the unbound antibodies, suspended in washing buffer, and analyzed using the BD Accuri (BD Biosciences). The acquisition was made in BD-Accuri C6 Software, and the cytokine concentrations determined using the FCAP Software v.3.0 (BD, Biosciences). The limit of detection was 0.1 pg/mL for IL-2, 0.03 pg/mL for IL-4, 1.4 pg/mL for IL-6, 0.5 pg/mL for IFN-gamma, 0.9 pg/mL for TNF-alpha, 0.8 pg/mL for IL-17A, and 16.8 pg/mL for IL-10.
10
Cytokine Analysis in Saliva using CBA
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The analysis of cytokines in saliva was performed using a CBA Cytokine Inflammatory Kit (Becton Dickinson, USA) to detect IL-1β, IL-6, IL-8, IL-10, IL-12p70 and TNF-α. All analyses were performed in duplicate.
In brief, 25 μl of fluorescent particles conjugated to antibodies specific to each cytokine were added to 25 μl of the saliva and incubated for one hour at room temperature, away from light. Then, 25 μl of the secondary antibody conjugated to a fluorochrome was added to the mixture and incubated for two hours at room temperature. The results were compared to a standard curve with serially diluted cytokines. The particles were washed to remove the unbound antibodies, resuspended in wash buffer and analyzed using a BD Accuri (BD Biosciences). Data acquisition was performed using BD Accuri C6 Software, and concentrations were determined using FCAP Software v.3.0 (BD Biosciences).
In brief, 25 μl of fluorescent particles conjugated to antibodies specific to each cytokine were added to 25 μl of the saliva and incubated for one hour at room temperature, away from light. Then, 25 μl of the secondary antibody conjugated to a fluorochrome was added to the mixture and incubated for two hours at room temperature. The results were compared to a standard curve with serially diluted cytokines. The particles were washed to remove the unbound antibodies, resuspended in wash buffer and analyzed using a BD Accuri (BD Biosciences). Data acquisition was performed using BD Accuri C6 Software, and concentrations were determined using FCAP Software v.3.0 (BD Biosciences).
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