| Immunostaining of Formaldehyde-fixed Metaphase Chromosome from Untreated and Aphidicolin-treated DT40 Cells
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Author:
Date:
2017-05-05
[Abstract] During mitosis chromosomes are condensed into dense X-shaped structures that allow for microscopic determination of karyotype as well as inspection of chromosome morphology.
This protocol describes a method to perform immunostaining of formaldehyde-fixed metaphase chromosomes from the avian cell line DT40. It was developed to characterize the localization of YFP-tagged TopBP1 on mitotic chromosomes and specifically determine the percentage of TopBP1 foci that formed on breaks/gaps as well as ends of individual metaphase macrochromosomes (Pedersen et al., 2015). For this purpose immunostaining of YFP was applied. However, the protocol may be optimized for other cell lines or epitopes.
[摘要] 在有丝分裂期间，染色体被浓缩成致密X型结构，允许显微检测染色体核型和检查染色体形态。
该方案描述了从禽细胞系DT40进行甲醛固定的中期染色体的免疫染色的方法。 它被开发用于表征YFP标记的TopBP1在有丝分裂染色体上的定位，并且具体确定形成于断裂/间隙以及单个中期大染色体末端的TopBP1灶的百分比（Pedersen等，2015）。 为此，应用YFP的免疫染色。 然而，该协议可以针对其他细胞系或表位进行优化。
【背景】染色中期染色体的显微镜分析是经典的细胞遗传学技术，广泛用于研究和诊断。基本原理包括通过锭子不稳定试剂如谷氨酸诱导中期细胞周期停滞，这将触发主轴装配检查点，从而阻止细胞在中期。这用于富集浓缩染色体的细胞。随后将细胞在低渗溶液中进行溶胀，然后在显微镜载玻片上铺展有丝分裂细胞。最终结果是来自单细胞的显微镜检测染色体，便于染色体核型分析以及个体染色体的研究。传统上，肿胀的细胞用甲醇和乙酸（3:1）固定，然后铺展在载玻片上（Hungerford，1965; Ronne等，1979）。
这里描述的方法使用甲醛而不是甲醇进行固定。这可以用于随后用与甲醇固定不相容的抗体染色。该方案针对鸡DT40细胞的中期传播进行了优化，YFP标记的TopBP1在中期大染色体上的免疫染色（Pedersen等，2015）。 ...
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| MHC Class II Tetramer Labeling of Human Primary CD4+ T Cells from HIV Infected Patients
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Author:
Date:
2017-03-20
[Abstract] Major Histocompatibility Complex (MHC) tetramers have been used for two decades to detect, isolate and characterize T cells specific for various pathogens and tumor antigens. In the context of Human Immunodeficiency Virus (HIV) infection, antigen-specific CD8+ T cells have been extensively studied ex vivo, as they can be readily detected by HIV peptide-loaded MHC class I tetramers. In contrast, the detection of HIV-specific CD4+ T cells has proven more challenging, due to the intrinsically lower clonal expansion rates of CD4+ T cells, and to the preferential depletion of HIV-specific CD4+ T cells in the course of HIV infection.
In the following protocol, we describe a simple method that facilitates the identification of CD4+ ...
[摘要] 主要组织相容性复合物（MHC）四聚体已经使用二十年来检测，分离和表征各种病原体和肿瘤抗原特异性的T细胞。在人类免疫缺陷病毒（HIV）感染的背景下，抗原特异性CD8 +细胞已经在体外广泛研究，因为它们可以容易地被HIV肽 - 加载MHC I类四聚体。相比之下，HIV特异性CD4 + sup + T细胞的检测已被证明更具挑战性，因为CD4 + sup + T细胞的本质上较低的克隆扩增率以及优先艾滋病毒感染过程中艾滋病毒特异性CD4 + T细胞的消耗。＆nbsp;在以下协议中，我们描述了一种简单的方法，该方法有助于使用肽负载的MHC II类四聚体来鉴定HIV-1衣壳表位特异性的CD4 + / T细胞。可以分析四聚体标记的CD4 T细胞的细胞表面表型和/或FACS分选用于进一步的下游应用。成功检测特异性CD4 + / / T细胞离体的关键是选择导致高亲和力T细胞受体（TCR）的肽/ MHC II组合， （Benati等人，2016）。 MHC II四聚体阳性细胞的可靠检测的第二个关键点是系统地使用负载无关肽的对照四聚体，样品和对照管在相同的条件下进行处理。背景 在用抗PE微珠对四聚体-PE标记的细胞进行磁力富集后，在纯化的CD4 + T细胞中检测到罕见的HIV特异性MHC II四聚体阳性细胞（Seth等人， em>。，2005）。我们发现使用经验证的肽/ MHC ...
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| Protocol for T-cell Adhesion Strength on Tumor Cells under Flow Conditions
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Author:
Date:
2013-10-20
[Abstract] This method allows evaluating the relative adhesion strength between T lymphocytes and specific adherent target cells using a shear force in flow chambers. It is based on the measure of the resistance of conjugates formed between T cells and adherent tumor cells to shear stress in a microfluidic system. For this purpose, T cells, stained with a CellTracker probe, are added into flow channels containing a monolayer of adherent target cells and their progressive detachment under a constant shear stress is then recorded using a fluorescent microscope.
[摘要] 该方法允许使用流动室中的剪切力评估T淋巴细胞和特异性粘附靶细胞之间的相对粘附强度。 其基于在微流体系统中T细胞和粘附的肿瘤细胞之间形成的缀合物对剪切应力的抗性的测量。 为此，将用CellTracker探针染色的T细胞加入含有单层粘附靶细胞的流动通道中，然后使用荧光显微镜记录在恒定剪切应力下的其逐渐分离。
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