This assay evaluated the stimulating effect of DCs on T cells. DCs cocultured with TSLP 24 h were used as stimulator cells. T lymphocytes were obtained from C57/BL6 mice using the lymphocyte separation medium (Ficoll-Paque PLUS, GE, MA, USA). The stimulated DCs and T cells were cocultured in 6-well flat-bottom plates at a ratio of 1 : 10 in the RPMI-1640 medium with 10% FBS as described previously [25 (link)]. The different concentrations of YPF were added in the mixed lymphocyte for 48 h. The collected cells were detected by flow cytometry for CD4 and IL-13 (eBioscience, Grand Island, NY, USA). The collected culture supernatants were used for cytokine level assessment with CBA flex kits (BD Biosciences, San Jose, California, USA).
Cba flex kit
Manufactured by BD
Sourced in United States
The CBA flex kit is a laboratory equipment product that provides a flexible solution for automated clinical chemistry analysis. It offers a modular design to accommodate various configurations and sample throughput requirements.
Automatically generated - may contain errors
Lab products found in correlation
Facscanto 2 flow cytometer, by BD (2 mentions)
Ficoll paque plus, by GE Healthcare (1 mentions)
Facsarray, by BD (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Sodium dodecyl sulfate (sds), by Merck Group (1 mentions)
Dimethylformamide, by Merck Group (1 mentions)
Fcap array software version 3, by BD (1 mentions)
Lsrii flow cytometer, by BD (1 mentions)
7 protocols using cba flex kit
1
Evaluating DC-Induced T Cell Activation
2
Knee Joint Cytokine and Chemokine Levels
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The concentration of IL-1β, -6, -17A and (C-X-C) ligand (CXCL)1/KC in the supernatant of knee joint washes was determined by using CBA Flex Kits, according to the manufacturer’s instructions (BD, Franklin Lakes, NJ, USA). Data were acquired in a FACSCanto II flow cytometer (BD) and analyzed with the program FCAP Array v3.0 (Soft Flow Inc., Pecs, Hungary). Detection limits for IL-1β, IL-6, IL-17A and CXCL1/KC were 4.32, 9.01, 8.72 and 10.48 pg/mL, respectively.
3
Cytokine Profiling of Th1 and Th2 Responses
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A comprehensive panel of Th1 and Th2 cytokines (IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, IFN-γ, TNF) was assayed using the mouse cytometric bead array (CBA) flex kit (BD Biosciences, San Jose, CA) also as previously described32 (link), with 5 × 105 splenocytes/well and 1.5 × 105 peptide-pulsed or antigen-transfected A20 APCs/well in 96-well round-bottom plates (for individual mice) and a 48 hr incubation period. Fluorescence intensity of the detection beads was measured on the FACSArray (BD Biosciences, San Jose, CA) and data analysed using FlowJo software (FlowJo software version 9.1 Treestar Inc.).
4
Secreted Factor Analysis via CBA Flex Kit
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Analysis of secreted factors in the supernatant was performed using the CBA Flex Kit (BD Biosciences, San Jose, CA, USA) according to manufacturer’s instructions.
5
Detailed Characterization of Recombinant Human Gelsolin
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Methods of expression, purification, and systematic characterization of rhuGSN were followed as described by us earlier [22 (link)–24 (link)]. Briefly, His-tag at N-terminal bearing gelsolin was expressed in E. coli in inducible format. Cells were lysed, and the protein was extracted from cytoplasm using a Ni-NTA-based affinity column followed by gel filtration. The purity and identity of protein were ascertained by expected migration in SDS-PAGE (followed by antigelsolin western blots) and MALDI-TOF, respectively. Furthermore, gelsolin was routinely characterized by its ability to depolymerize/nucleate F-actin and small angle X-ray scattering (SAXS) experiments in our group. Overall, purity, identity, and precise concentration of pGSN were done to required diligence before commencing experiments. Importantly, the protein was eluted through a Polymyxin B column to remove base levels of LPS to below the detection level before using samples for cell line experiments [22 (link)]. A total antioxidant capacity estimation kit (BioVision Inc., USA), CBA-Flex kit (BD Biosciences), DMEM (Genetix Biotech), Fetal Bovine Serum (FBS) (GIBCO), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Sigma), sodium dodecyl sulfate (Sigma), dimethylformamide (Sigma), and H2O2 (Merck) were used in the study.
6
Cytokine profiling of JEV-infected moDCs
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CBA was performed to quantitatively measure the cytokine level of IL-6, IL-8, IL-10, IL-12, MCP-1, RANTES, IFNγ and TNFα in JEV-infected moDCs using the CBA Flex kit from BD biosciences (Table S3 Figure S2
7
Quantifying Proinflammatory Cytokines in Hippocampus
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Proinflammatory cytokines, TNFα and IFNγ, were analyzed in hippocampal tissue lysates and quantified as per instructions provided with the CBA flex kit (BD Biosciences, U.S.A). The acquisition was done on LSR-II Flow Cytometer at BD FACS Academy, Jamia Hamdard, New Delhi, India and analysis was done using FCAP ARRAY software version 3.0 (BD Biosciences, U.S.A.). The levels of TNFα and IFNγ were quantified as pg/ml of tissue lysate.
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