Cryo-preserved peripheral blood mononuclear cells (PBMCs) were thawed and cultured overnight in 5% CO2 at 37°C with Roswell Park Memorial Institute (RPMI) media supplemented with 4% human serum type AB (Lonza), 1% penicillin/streptomycin, and 1% 200 mM L-glutamine. Following the overnight culture, IFN-γ production by T-cells was assessed using enzyme-linked immunospot assays of PBMCs (5×105 cells/well), as previously described[15 (link)]. PBMCs were stimulated either with a mixture of MeV hemagglutinin, fusion, and nucleoprotein proteins as 20 amino acid peptides (11 amino acids overlapping) at 1μg/mL or with a lysate from MeV-infected Vero cells (Advanced Biotechnologies) at 10μg/mL for 40 hours. RPMI media and Con A (5μg/mL) were used as negative and positive controls, respectively. After stimulation, the plates were incubated with biotin-conjugated antibodies to human IFN-γ, then developed and read, as previously described[15 (link)]. Low and positive T-cell responses were categorized as <20 and ≥20 spot-forming cells (s.f.c.)/million PBMCs, respectively.
Human serum type ab
Manufactured by Lonza
Human serum type AB is a laboratory reagent derived from the plasma of individuals with the AB blood type. It serves as a control or reference material in various immunological and biochemical assays that require human serum components.
Automatically generated - may contain errors
4 protocols using human serum type ab
1
Quantifying IFN-γ T-cell Responses to Measles Virus
2
Quantifying DENV Antigen in Infected PBMCs
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Peripheral blood mononuclear cells (PBMCs) were infected with DENV-4 (MOI 10) for 2 h and treated with the NTCs of the active compounds for five days at 37 °C and 5 % CO2. After incubation, cells were analyzed for DENV antigen quantification by FACS. Briefly, the cells were blocked with PBS, 5 % FCS (Gibco-Invitrogen, South America) and 1 % human serum type AB (Lonza, Walkersville, MD) for 20 min at room temperature. Following this, the cells were fixed using Cytofix/Cytoperm (Becton & Dickinson, San Jose, CA), washed using Perm/Wash, and stained with the 4G2 monoclonal (specific for flavivirus envelope protein) for 20 min at 37 °C. After incubation, the cells were washed with Perm/Wash and incubated for 20 min at 37 °C with the secondary antibody (donkey anti-mouse conjugated with Alexa-488; Life Technologies). Finally, cells were washed twice with Perm/Wash and analyzed using a FACSCanto II (BD Biosciences). Data were analyzed by two-way ANOVA followed by the Bonferroni post test.
3
Dengue and Zika Serum Neutralization
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Serum samples were collected from confirmed dengue and Zika patients who resided in Ha Noi, northern Viet Nam, and in Central Vietnam between 2015–2018. A commercially available serum sample, obtained from a healthy donor (Human Serum Type AB, Lonza), was used as negative control serum. Serum samples were heat-inactivated at 56 °C for 30 min before use in the experiments. All sera used had been tested for DENV or ZIKV antibodies by IgG ELISA and IgM ELISA. Mouse monoclonal antibodies (200 µg/mL) that were flavivirus cross-reactive (IgG2a: MAb HB-112 D1-4G2-4-15 and 6B6C-1) and DENV-2 serotype-specific (IgG1: Mab HB-46 3H5-1) were also used for the neutralization assay.
4
Pyrene-modified TBA Nuclease Stability
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The chemical stability of pyrene-modified TBA was analyzed in the presence of 80% (v/v) human serum type AB (Lonza), which contains various nucleases such as endonucleases and exonucleases. The degradation of 5 μM pyrene-modified TBA by nucleases present in the serum was examined after various incubation times at 37°C. Reactions were quenched at chosen times by the addition of a 5-fold excess volume of stop solution (80 wt% formamide, 10 mM Na2EDTA and 0.1% blue dextran) and incubated at 95°C for 15 min. Before the denaturing PAGE, the precipitate of proteins in the serum was removed by centrifugation. Gels were run at 250 V for 100 min at 25°C and stained with SYBR® Gold (PerkinElmer Life Sciences) and imaged using a fluorescent imager (Fujifilm, FLA-5100).
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