Improvement and application of intracellular cytokines staining assay to detect SIVmac239-specific cellular immunity in SIV-infected rhesus monkeys
  
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DOI:10.3969/j.issn.1005-4847.2012.01.005
KeyWord:SIV;Specific cellular immunity;Intracellular cytokines staining;Detection, optimization; Rhesus monkey
                 
AuthorInstitution
吴芳新 中国医学科学院医学实验动物研究所,卫生部人类疾病比较医学重点实验室,国家中医药管理局人类疾病动物模型三级实验室,北京
王卫 中国医学科学院医学实验动物研究所,卫生部人类疾病比较医学重点实验室,国家中医药管理局人类疾病动物模型三级实验室,北京
丛喆 中国医学科学院医学实验动物研究所,卫生部人类疾病比较医学重点实验室,国家中医药管理局人类疾病动物模型三级实验室,北京
刘克剑 中国医学科学院医学实验动物研究所,卫生部人类疾病比较医学重点实验室,国家中医药管理局人类疾病动物模型三级实验室,北京
熊竞 中国医学科学院医学实验动物研究所,卫生部人类疾病比较医学重点实验室,国家中医药管理局人类疾病动物模型三级实验室,北京
魏强 中国医学科学院医学实验动物研究所,卫生部人类疾病比较医学重点实验室,国家中医药管理局人类疾病动物模型三级实验室,北京
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Abstract:
      ObjectiveTo improve the intracellular cytokines staining (ICS) assay and serve the detection of SIV-specific cellular immunity in SIV-infected rhesus monkeys.MethodsThree polyclonal activators were used for PBMC stimulation, and the best positive stimulators and the reasonable stimulating time were tested and determined. Five concentrations of SIVmac239 mixed peptides pool were designed to stimulate PBMC from SIV-infected rhesus monkeys, followed by culturing in vitro and detected with flow cytometry at different times, and the optimal concentration of SIV mixed peptides pool and the best stimulating time were ascertained finally. Then, this method was used for detection of the SIV-specific cellular immunity in the monkeys. ResultsCombination of PMA+ ionomycin could be used as the best positive stimulator. Cytokines secreted from T cells were detected at the highest level by 2 μg/mL SIVmac239 mixed peptides pool at 37℃ and in 5% CO2 culturing for 16 h in vitro. ConclusionsOptimization of the experimental conditions of ICS has been successfully accomplished, and it may be useful for pre-clinical evaluation of AIDS drugs and vaccine research.
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