To analyze cytokine production, lung lymphocytes were collected from three mice per group at 10 days after the fifth immunization. Lung lymphocytes (5−10×105) were added to each well of a 24-well plate and stimulated with S-RBD protein (1 µg/well) for 1 or 2 days. The culture medium was collected after stimulation; expression levels of mouse IL-10, IFN-γ, TNF-α, and IL-17A were quantified using a Cytometric Bead Array Kit (BD Biosciences, Franklin Lakes, NJ, USA), in accordance with the manufacturer’s instructions. Briefly, Ab-coated beads for each cytokine were mixed and incubated with culture medium, then incubated for 2 h at room temperature with PE-conjugated detection Abs. After the beads had been washed, flow cytometric analyses were performed using a Cytoflex Flow Cytometer (Beckman Coulter); data analyses were performed using FCAP Array™ software (BD Biosciences). Cytokine concentrations were calculated using a standard curve that had been generated from cytokine standards.
Cytometric bead array kit
Manufactured by BD
Sourced in United States
The Cytometric bead array kit is a multiplex assay that allows for the simultaneous detection and quantification of multiple analytes in a single sample. The kit utilizes beads coated with capture antibodies that bind to the target analytes, which are then detected using fluorescent-labeled detection antibodies.
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Fcap array software, by BD (12 mentions)
Facscanto 2, by BD (9 mentions)
Facscalibur flow cytometer, by BD (8 mentions)
Facscanto 2 flow cytometer, by BD (6 mentions)
Fcap array software version 3, by BD (4 mentions)
Lsrfortessa, by BD (4 mentions)
Accuri c6 plus, by BD (4 mentions)
Facscalibur, by BD (4 mentions)
Fcap array v3, by BD (4 mentions)
Facscalibur ow cytometer, by BD (3 mentions)
Cytofix cytoperm plus kit, by BD (3 mentions)
Mouse il 1β elisa kit, by R&D Systems (2 mentions)
Bullet blender, by Next Advance (2 mentions)
1 fluoro 2 4 dinitrofluorobenzene, by Merck Group (2 mentions)
Macs technology, by Miltenyi Biotec (2 mentions)
Golgistop, by BD (2 mentions)
Facsaria, by BD (2 mentions)
Facscanto, by BD (2 mentions)
Ready set go elisa kit, by Thermo Fisher Scientific (2 mentions)
Facsverse, by BD (2 mentions)
157 protocols using cytometric bead array kit
1
Quantifying Cytokine Profiles in Lung Lymphocytes
2
Cytokine Quantification in oxLDL-Treated Cells
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After cells were incubated with oxLDL for 24 hrs, culture media were collected to measure the protein levels of soluble cytokines. The concentrations of TNF-α and MCP-1 were determined by LSRFortessa flow cytometry (BD Biosciences, San Jose, CA) using a Cytometric Bead Array kit (BD PharMingen, Franklin Lakes, NJ) as per manufacturer’s instruction. Individual sample (50 μl/each) was tested in duplicate. The concentrations of cytokines were quantified by CellQuest Pro using a CBA software. The concentration of IL-1β was measured by ELISA using a mouse IL-1β ELISA kit (R&D System, Minneapolis, MN) as per manufacturer’s instruction.
3
Quantifying Serum Cytokine Levels
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Peripheral blood was collected in Gel BD SST II Advance tubes (BD Biosciences, CA, USA), and serum samples were isolated by centrifugation at 1.372× g for 5 min at 25 °C. IL-4 and IL-10 concentrations were detected by flow cytometer FACSCanto II (BD Biosciences, CA, USA), using the cytometric bead array kit (BD Biosciences, CA, USA). The analyses were performed by using BDFCAP array software and data were presented as pg/mL.
4
Cytokine Profiling of Splenocyte Responses
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Splenocytes were prepared and counted from each mouse in different groups as described previously (32 (link)). Subcomponent-specific cytokines Th1 (TNF-α and IFN-γ), Th2 (IL-4), and Th17 (IL-17A) were detected using cytometric bead array (CBA). 5 × 106 cells were plated into each well of a 24-well plate and incubated with RPMI1640 medium (negative control), or RPMI1640 medium with specific proteins including Rv0577, Rv2875, Rv3044, or Rv2073c (each 10 µg/mL), respectively. The supernatant was collected 24 h later and Cytometric Bead Array kit (BD Biosciences, NJ, USA) was performed according to the manufacturer’s instructions.
Alternatively, 5 × 106 cells were incubated with CMFO protein (10 µg/mL) at 37°C and 5% CO2. RPMI1640 medium was used as a negative control and PPD (10 mg/mL, Statens Serum Institut, Denmark) was set as a positive control. After an incubation of 72 h, the supernatant was collected and a commercial mouse IFN-γ ELISA kit (Multi Sciences, Hangzhou, China) were used to detect the concentration of IFN-γ (20 (link)–22 (link)). The results are expressed as mean ± SD (pg/mL) for each group (n = 6).
Alternatively, 5 × 106 cells were incubated with CMFO protein (10 µg/mL) at 37°C and 5% CO2. RPMI1640 medium was used as a negative control and PPD (10 mg/mL, Statens Serum Institut, Denmark) was set as a positive control. After an incubation of 72 h, the supernatant was collected and a commercial mouse IFN-γ ELISA kit (Multi Sciences, Hangzhou, China) were used to detect the concentration of IFN-γ (20 (link)–22 (link)). The results are expressed as mean ± SD (pg/mL) for each group (n = 6).
5
CAR-T Cell Cytokine Secretion Assay
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Co-culture target cells and CAR-T cells or CAR-T cells only at a 1:1 ratio in basal XSFM (repeat activation assay co-culture, steps 31–34).
For each well, transfer cells to sterile tube and centrifuge at 300
Collect supernatant and store at −80°C for cytokine analysis or proceed directly to step 40
Perform flow cytometry quantification of TNF-α and IFN-γ cytokines using Cytometric Bead Array Kit (BD Biosciences). For a detailed protocol, please refer to the BD™ Cytometric Bead Array (CBA) Human Th1/Th2 Cytokine Kit Instruction Manual (BD Biosciences, 2019 ). Briefly, supernatant or provided cytokine standard solutions are serially diluted and cytokine bead suspensions are added. Beads are then washed and used in flow cytometry to quantify presence of TNF-α and IFN-γ cytokines.
6
Quantifying Viral Burdens and Immune Responses
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For measuring viral burdens in organs, spleens and livers from infected mice were homogenized by Magna lyser (Roche Applied Science) and the lysates were appropriately diluted and overlaid on mouse embryonic fibroblasts cells in duplicates in 10% Dulbecco Modified Eagle’s Medium (DMEM) (DMEM from HyClone, 10% FCS, 10 mM HEPES, 1× penicillin/streptomycin, 1% l -Glutamine). Plaques were counted 3 days later and represented as log PFU/organ. Blood serum and lysates of homogenized spleen and livers from mice were appropriately diluted and analyzed for the production of mouse IFN-γ using Cytometric Bead Array kit (BD Biosciences). Samples were prepared according to manufacturer’s instructions, acquired on FACS Cyan ADP (Beckman Coulter) and analyzed using the BD FCAP Array Software (BD Biosciences).
7
Macrophage Cytokine Response to LPS
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RAW 264.7 macrophages were cultured in RPMI-1640 medium supplemented with 10% foetal bovine serum. The cells (1 × 104) were plated in 96-well flat-bottom plates (Corning Costar, Corning, NY) and incubated in the presence of E. coli LPS or Bacteroides LPS for 12, 24, 48, and 72 h at 37 °C with 5% CO2. The cytokine levels were analysed using the Cytometric Bead Array Kit (BD Biosciences, San Diego, CA, USA), according to the manufacturer’s instructions.
8
Quantifying In Vivo Viral Propagation and Inflammation
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To measure ApoBD-mediated viral propagation in vivo, B6 mice with treated with either 1000 pfu PR8 or FACS-isolated ApoBDs (0.4–1 × 105) as described above. Mice were sacrificed on day 3 post treatment, and lung tissue was harvested and homogenised. Lung viral titres in the lungs were determined using plaque assays on MDCK cells as previously described51 (link). Inflammatory cytokine release were quantified on lung homogenates using a cytometric bead array kit (BD Biosciences) according to the manufacturer’s instructions and as previously described51 (link).
9
Quantification of Serum Immunoglobulins Using Flow Cytometry
10
Cytokine Production by CD4+ T Cells
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Highly purified CD4+ T cells were isolated as described above. Purified CD4+ T cells (1 × 105) were co-cultured with 1 × 104 irradiated DCs (30 Gy). Recombinant murine anti-IL-12p70 or/and anti-INF-γ (R&D Systems, USA) at a concentration of 10 ng/ml were added to T cell cultures that have been stimulated with DCs following exposure to SOCS1 siRNA. After 48 h, cells were activated with phorbol myristate acetate (50 ng/ml) and ionomycin (50 ng/ml) in the presence of brefeldin A (10 µg/ml) for 6 h. For intracellular cytokine production analysis, cells were harvested, washed, fixed, and permeabilized using the CytoStain kit (BD Pharmingen, USA) according to the manufacturer’s instructions. Cells were then stained with 0.5 µg/test of anti-murine IL-4-PE, anti-murine-IFN-γ FITC, and anti-murine IL-17 (BD Pharmingen, USA) and were analyzed by flow cytometry. IFN-γ, IL-4, and IL-17 production by CD4+ T cells was also assessed using the Cytometric Bead Array kit from BD Pharmingen.
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