The chemokines CCL2, CXCL8, CXCL9, and CXCL10 were quantified in serum using the CBA method with the commercial CBA Human Chemokine Kit (BD Pharmingen, San Diego, CA, USA), following the manufacturer’s instructions. Analysis was performed on a flow cytometer (LSR Fortessa–BD Biosciences, San Diego, CA, USA), and data analysis was conducted using the FCAP Array 3.0 software (BD Biosciences, San Diego, CA, USA). The detection limits were 2.7 pg/mL (CCL2), 0.2 pg/mL (CXCL8), 2.5 pg/mL (CXCL9), and 2.8 pg/mL (CXCL10).
Cba human chemokine kit
Manufactured by BD
Sourced in United States
The CBA Human Chemokine Kit is a multiplex assay designed to quantify multiple chemokines in biological samples. It utilizes flow cytometry technology to provide reliable and accurate measurements of chemokine levels.
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18 protocols using cba human chemokine kit
1
Multiplex Chemokine Quantification in Serum
2
Quantification of Chemokine and GM-CSF Secretion in A375 Cells
3
Chemokine Quantification using CBA
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Chemokine quantification was performed using CBA – Human Chemokine Kit (BD Biosciences, USA), following the manufacturer’s instructions in culture supernatants. Briefly, 50 µl of capture beads for MCP1/CCL2, RANTES/CCL5, IL-8/CXCL8, MIG/CXCL9, and IP10/CXCL10, 50 µl of Detection Reagent and 50 µl of the studied sample or standard were added consecutively to each sample tube and incubated for 3 h at room temperature, in the dark. Next, the samples were washed with 1 ml of wash buffer and centrifuged. After discarding the supernatant, the pellet was resuspended in 300 µl buffer and analyzed in a FACS LSR Fortessa flow cytometer (BD Biosciences, USA) (Souza et al., 2021 (link)). Raw data were then analyzed using FCAP Array software (BD Biosciences, USA). The detection limits of each chemokine were as follows: 2.7 pg/mL for MCP1/CCL2, 1.0 pg/mL for RANTES/CCL5, 0.2 pg/mL for IL-8/CXCL8, 2.5 pg/mL for MIG/CXCL9 and 2.8 pg/mL for IP10/CXCL10.
4
Quantitative Chemokine Profiling of Cell Cultures
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Chemokine quantification in culture supernatants was performed using CBA – Human Chemokine Kit (BD Biosciences, USA) in accordance with manufacturer’s instructions. Briefly, 50 µl of capture beads for MCP1/CCL2, RANTES/CCL5, IL-8/CXCL8, MIG/CXCL9, and IP10/CXCL10, 50 µl of Detection Reagent, and 50 µl of the studied sample or standard were added consecutively to each sample tube and incubated for 3 h at room temperature, in the dark. Next, the samples were washed with 1 ml of Wash buffer, and centrifuged. After discarding the supernatant, the pellet was resuspended in 300 µl buffer and analyzed in a FACS LSR Fortessa flow cytometer (BD Biosciences, USA). Raw data was then analyzed using FCAP Array software (BD Biosciences, USA). The detection limits of each chemokine were as follows: 2.7 pg/ml for MCP1/CCL2, 1.0 pg/ml for RANTES/CCL5, 0.2 pg/ml for IL-8/CXCL8, 2.5 pg/ml for MIG/CXCL9, and 2.8 pg/ml for IP10/CXCL10.
5
Quantification of Chemokine and GM-CSF Secretion in A375 Cells
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A375 cell production of chemokine proteins was quantified using a CBA Human Chemokine Kit (BD Biosciences Cat #552990) and GM-CSF production was measured using Human GM-CSF ELISA Kit (Abcam Cat #ab174448). In brief, A375 cells transduced with Cas9 and non-targeting sgRNA or BIRC2-targeting sgRNAs were plated at 4 × 105 in a 6-well dish and cultured for 24 h. Media supernatant was collected and spun at 1500rpm for 10 min to pellet cells and debris. Supernatant was collected for analysis. In some cases, media was diluted 1:5 using assay diluent to ensure detected values were within standard calibration curves.
6
Evaluating Complement Activation in Plasma
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The generation of the soluble C5b-9 complex (sTCC) in plasma samples collected from human whole blood assays was evaluated by enzyme-linked immunoassay (ELISA) using the MicroVue SC5b-9 Plus EIA kit (Quidel Corporation, San Diego, CA, USA). Plasma levels of C3a/C3a-desArg, C4a/C4a-desArg, and C5a/C5a-desArg were determined using the BD Cytometric Bead Array (CBA) Human Anaphylatoxin Kit (BD Biosciences, San Jose, CA, USA). The measurements of cytokines (IL-2, IL-4, IL-6, IL-10, IL-17A, IFN-γ, and TNF) and chemokines (CXCL8/IL-8, CCL5/RANTES, CXCL9/MIG, CCL2/MCP-1 and CXCL10/IP-10) were performed using a BD cytometric bead array (CBA) Th1/Th2/Th17 Human cytokine kit (BD Biosciences) and CBA Human Chemokine Kit (BD Biosciences), respectively. All assays were performed according to the manufacturers’ instructions.
7
Multiplex Cytokine and Chemokine Quantification
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A Cytometric Bead Array (CBA) Human Inflammatory Cytokines Kit (BD Biosciences) was employed to quantify plasma concentrations of IL-1β, IL-6, IL-10, IL-12p70, and TNF, while CCL2/MPC-1, CCL5/RANTES, CXCL8/IL-8, CXCL9/MIG, and CXCL10/IP-10 quantification was performed using a CBA Human Chemokine Kit (BD Biosciences), each following the manufacturer’s instructions. Data acquisition and analysis were performed using a FACSArray Bioanalyzer (BD Biosciences) and FlowJo v10.0.5 (Tree Star) software, respectively.
8
Immune Modulation by B7-H4 in T Cell Activation
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CHO/B7-H4 cells (2 × 10 4 /well) were pre-incubated into 96-well plates overnight. Peripheral blood mononuclear cells (PBMCs, 1 × 10 6 /well) isolated from healthy controls were added with anti-CD3 mAb (100 ng/ml) and B7-H4 mAbs (1 µg/ml or 10 µg/ml) or anti-CD3 mAb plus isotype control antibody (mouse IgG, 10 µg/ml) at 37°C. After 72 h in co-culture, cell proliferation was investigated by cell counting kit 8 (CCK-8) (Mashiki-machi, Kiyushu, Japan). Meanwhile, supernatants were collected and cytokine concentrations were analyzed using a cytometric bead array (CBA) human chemokine kit (BD Biosciences, San Jose, CA, USA). The CBA kit can be used to quantitatively measure interleukin (IL)-2, IL-4, IL-6, IL-10, IL-17, tumor necrosis factor (TNF)-α and interferon (IFN)-γ levels in a single sample.
9
Comprehensive Cytokine and Cellular Analysis
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Cytokine and chemokine assessments. The BD™ Cytometric Bead Array (CBA) Human Inflammatory Cytokines Kit was used to quantitatively measure IL-8, IL-1β, IL-6, IL-10, TNF and IL-12P protein levels in a single sample.
BD™ CBA Human Chemokine Kit was used to quantitatively measure IL-8 (CXCL8/IL-8), RANTES (CCL5/RANTES), monokine induced by IFN-γ (CXCL9/MIG) and IFN-γ-induced protein-10 (CXCL10/IP-10) levels in a single sample. Cell subtype assessments. BD™ Multitest IMK kits were used with CD3/CD8/CD45/CD4 reagent and CD3/CD16 + CD56/CD45/ CD19 reagent. Flow cytometry analysis measures the emission of optical signals, after passing through a laser beam. In our investigation a "lyse -no wash" method was used (35) . The measurement was performed using CellQuest Protocol according to the guidelines of the manufacturer on a BD™ FascCalibur. Assessment of cortisol levels. Serum for analysis was obtained between 07.40 h and 10.15 h. Cortisol levels were analyzed using Siemens ADVIA Centaur ® cortisol assay, a competitive immunoassay using direct chemiluminescent technology.
BD™ CBA Human Chemokine Kit was used to quantitatively measure IL-8 (CXCL8/IL-8), RANTES (CCL5/RANTES), monokine induced by IFN-γ (CXCL9/MIG) and IFN-γ-induced protein-10 (CXCL10/IP-10) levels in a single sample. Cell subtype assessments. BD™ Multitest IMK kits were used with CD3/CD8/CD45/CD4 reagent and CD3/CD16 + CD56/CD45/ CD19 reagent. Flow cytometry analysis measures the emission of optical signals, after passing through a laser beam. In our investigation a "lyse -no wash" method was used (35) . The measurement was performed using CellQuest Protocol according to the guidelines of the manufacturer on a BD™ FascCalibur. Assessment of cortisol levels. Serum for analysis was obtained between 07.40 h and 10.15 h. Cortisol levels were analyzed using Siemens ADVIA Centaur ® cortisol assay, a competitive immunoassay using direct chemiluminescent technology.
10
Cytokine and Chemokine Profiling by Bead Array
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Selected inflammatory cytokines in plasma were determined using the Cytometric Bead Array Human Inflammatory Cytokines kit (#551811, BD Biosciences, Becton and Dickinson, San Diego, CA, USA), following the manufacturer’s instructions. The kit is designated for quantitative and simultaneous measurement of interleukin (IL)-6, IL-8, IL-10, IL-12, and tumor necrosis factor-alpha (TNF-α). The BD CBA Human Chemokine Kit was used to evaluate selected chemokine levels (pg/mL) (552990, BD Biosciences, Becton and Dickinson, San Diego, CA, USA). The kit is designated for the quantitative and simultaneous measurement of IL-8, RANTES, monokine induced by interferon γ (CXCL9/MIG), monocyte chemoattractant protein-1 (CCL2/MCP-1), and interferon γ-induced protein-10 (CXCL10/IP-10). Data acquisition (300 events for each cytokine and chemokine) was performed using a BD FACSCanto II flow cytometer with BD FACSDiva Software and FCAP Array software, version 3.0 (BD Biosciences, San Jose, CA, USA).
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