The generation of the anaphylatoxins (C3a/C3a-desArg, C4a/C4a-desArg and C5a/C5a-desArg) was determined using the Human Anaphylatoxin Cytometric Bead Array-CBA (BD Biosciences Pharmingen, San Diego, CA, USA), following the manufacturer’s instructions. Cytometric analysis was performed using a FACSCanto II (Becton Dickinson, San Diego, CA, USA), and the data were analyzed using the Flow Cytometric Analysis Program (FCAP) Array 3.0 (Becton Dickinson, San Diego, CA, USA). Anaphylatoxins concentrations (µg/mL) were determined by linear regression from the standard curve. The generation of sTCC (SC5b-9) was determined using the MicroVue SC5b-9 Plus EIA Kit (Quidel Corporation, San Diego, CA, USA), according to the manufacturer’s instructions. The concentration of sTCC (µg/mL) in the samples was calculated from a linear regression of the standard curve.
Fcap array 3
Manufactured by BD
Sourced in United States
The FCAP Array 3.0 software is a data analysis tool designed for flow cytometry applications. It provides a platform for managing, analyzing, and visualizing flow cytometry data. The software supports a range of features, including data import, gating, and statistical analysis.
Automatically generated - may contain errors
Lab products found in correlation
Facscanto 2, by BD (9 mentions)
Lsrfortessa cell analyzer, by BD (4 mentions)
Lsrfortessa flow cytometer, by BD (3 mentions)
Human anaphylatoxin cytometric bead array cba, by BD (2 mentions)
Microvue sc5b 9 plus eia kit, by Quidel (2 mentions)
Facsverse flow cytometer, by BD (2 mentions)
Facsverse, by BD (2 mentions)
Human th1 th2 th17 cytometric bead array kit, by BD (2 mentions)
Facscanto 2 cytometer, by BD (2 mentions)
Cba mouse th1 th2 th17 cytokine kit, by BD (2 mentions)
Cba mouse inflammation kit, by BD (2 mentions)
Cytometric bead array cba human th1 th2 kit, by BD (2 mentions)
Cytometric bead array mouse th1 th2 th17 cytokine kit, by BD (2 mentions)
Facs verse flow cytometer, by Beckman Coulter (1 mentions)
Human chemokines kit, by BD (1 mentions)
Human inflammatory cytokine kit, by BD (1 mentions)
Human chemokine kit, by BD (1 mentions)
Human th1 th2 th17 kit, by BD (1 mentions)
Cytometric bead array, by BD (1 mentions)
Facscalibur flow cytometry equipment, by BD (1 mentions)
38 protocols using fcap array 3
1
Anaphylatoxins and sTCC Quantification
2
Anaphylatoxin and TCC Quantification
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C3a/C3a-desArg, C4a/C4a-desArg, and C5a/C5a-desArg were measured using the Human Anaphylatoxin Cytometric Bead Array (CBA) (BD Biosciences Pharmingen, San Diego, CA, USA), following the manufacturer's instructions. Cytometric analysis was performed using a FACSCanto-II (Becton Dickinson, San Diego, CA, USA), and the data were analysed with the Flow Cytometric Analysis Program (FCAP) Array 3.0 (Becton Dickinson, San Diego, CA, USA). Anaphylatoxin concentrations (μg/mL) were determined by linear regression from the standard curve. TCC (in its soluble form SC5b-9) was determined using the MicroVue™ SC5b-9 Plus EIA Kit (Quidel Corporation, San Diego, CA, USA), according to the manufacturer's instructions. TCC concentration (μg/mL) in the samples was calculated from a linear regression of the standard curve.
3
Quantifying Chemokine Levels in Syphilis
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All serum of seroresistant syphilitic patients and healthy controls were collected by sterile tubes containing separating glue with centrifuging at 900 g for 5 min and stored at −80℃ for subsequent use. The concentrations of serum chemokines were quantitatively measured by Cytometric Bead Array (CBA) Human Chemokine Kit(552990)comprising microbeads coupled with monoclonal antibodies (MoAb) against CXCL8, CCL2, CXCL9, CCL5, and CXCL10. The levels of the corresponding chemokines were acquired using a FACS Verse flow cytometer (Beckman Coulter, Inc.) according to the manufacturer's instructions (Beckman Coulter, Inc.) and analyzed by BD FCAP Array 3.0 software (Becton Dickinson and Company).
4
Cytokine Quantification in Serum
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Blood samples for the measurement of cytokines were collected early in the morning. The CBA assay (CBA immunoassay kit; Becton, Dickinson and Company) was used for quantitative determination of serum cytokines (Human Inflammatory Kit), according to the manufacturer's instructions. The Inflammatory CBA Kit comprises microbeads coupled to mAbs against IL-6, IL-10, TGF-β, IL-17A, and IL-23. A secondary phycoerythrin-labeled anti-cytokine antibody was used, and the concentration of individual cytokines was determined based on fluorescence intensity. Data were acquired on a FACSVerse flow cytometer (Becton, Dickinson and Company). Sample analysis was performed using BD FCAP Array 3.0 software (Becton, Dickinson and Company).
5
Serum IL-6 Measurement by Flow Cytometry
6
Quantitative Cytokine and Chemokine Profiling
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Blood samples for measurement of cytokines and chemokines were collected at the baseline survey in early morning. The Cytometric bread array assay (CBA immunoassay kit; Becton Dickinson, California) was used for the quantitative determination of the serum cytokines (Human Inflammatory kit) and chemokines (Human Chemokines kit) levels according to the instructions of the manufacturer. Data was acquired using a FACSVerse flow cytometer (Becton Dickinson, California). BD FCAP Array 3.0 software (Becton Dickinson, California) was used for sample analysis. The coefficients of variation intra and inter-assays were 5-10% and 7-12%, respectively. Based on their distributions, IL6, CXCL8, CCL2, CXCL9, CCL5 and CXCL10 were log-transformed and considered as continuous variables in our analysis. IL1β, IL10, IL-12 and TNF showed very low detectable levels and were considered as dichotomous variables.
7
Cytokine profiling of cell cultures
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The cell culture supernatants collected from the cell viability assay were centrifuged at 400× g and stored at −80 °C until use. The presence of cytokines and chemokines in the samples was assessed using ‘Cytometric Bead Arrays’ (CBA) with the ‘Human Chemokine Kit’, ‘Human Inflammatory Cytokine Kit’, and ‘Human Th1/Th2/Th17 Kit’ (BD Biosciences, Franklin Lakes, NJ, USA), following the manufacturer’s instructions.
In order to evaluate a potential direct cleavage of these cytokines and chemokines by B. lanceolatus venom, which could interfere with the analysis of cells supernatants, samples of cytokines and chemokines standard mixtures were incubated with the venom for 30 min at 37 °C in the presence or absence of proteases inhibitors (PMSF, EDTA, and 1,10-phenanthroline; 20 mM). The samples were subsequently submitted to ‘Cytometric Bead Arrays’, following the manufacturer’s instructions. Concentrations of cytokines and chemokines were calculated in pg/mL of supernatant from the standard linear regression curves, using FCAP Array 3.0 software (Becton Dickinson, San Jose, CA, USA).
In order to evaluate a potential direct cleavage of these cytokines and chemokines by B. lanceolatus venom, which could interfere with the analysis of cells supernatants, samples of cytokines and chemokines standard mixtures were incubated with the venom for 30 min at 37 °C in the presence or absence of proteases inhibitors (PMSF, EDTA, and 1,10-phenanthroline; 20 mM). The samples were subsequently submitted to ‘Cytometric Bead Arrays’, following the manufacturer’s instructions. Concentrations of cytokines and chemokines were calculated in pg/mL of supernatant from the standard linear regression curves, using FCAP Array 3.0 software (Becton Dickinson, San Jose, CA, USA).
8
Cytokine Secretion Measurement in PBMCs
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From the supernatants of human PBMC cultures, cytokine secretion (IFN-γ, TNF, IL-2, IL-4, IL-6, IL-10) was measured using the BD™ CBA Human Th1/Th2 Cytokine kit (Becton Dickinson, United States). The reading was performed in the FACSCalibur flow cytometry equipment (Becton Dickinson, United States), according to the manufacturer’s guidelines. The results were analyzed using the FCAP Array 3.0 software (Becton Dickinson, United States), being normalized with the results obtained by non-stimulated cells. So, if the value is presented as negative, it means that there is an inhibition of cytokine production since the detected value is lower than that presented by the non-stimulated PBMCs.
9
Cytokine Profiling in Lung Tumor Tissue
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Tumor tissue and peritumor tissue (100 mg) were lysed by Ultrasonic Sonicator (Virsonic 60). Cytokines IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-17A, TNF-α, IFN-γ, and IFN-α, were measured in lung homogenates supernatant using the human cytokines combined detection kit (Jiangxi Cell-Gene Biotech CO., LTD) and were analyzed by software FCAP Array 3.0 (BD Biosciences) after collection of events in a flow cytometer (Beckman Coulter).
10
Multiplex Cytokine Profiling in Pancreatic Tissue
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Pancreatic tissue from experimental groups was crushed with scissors and then processed with ultrasonic sonicator (Virsonic 60) in lysis buffer (50 mM Tris, 150 mM NaCl, 1 mM NaF, 0.2 mM Sodium orthovanadate, 200 mM Sodium Pirophosphate, 0.1% SDS, 1% TritonX-100) plus protease inhibitor cocktail. Cytokines IL-10, IL-17 A, TNF-α, IFN-γ, IL-6, IL-2 and IL-4 were measured in pancreas homogenates and 2D and 3D ASC conditioned medium (CM-2D and CM-3D) using the kit CBA mouse Th1/Th2/Th17 (BD cat. No. 560484). Samples were analyzed according to manual user guide and results were analyzed by software FCAP Array 3.0 (BD Biosciences) after collection of events in the flow cytometer (BD C6 Accuri).
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